Novel pharmaceutical composition comprising particles comprising a complex of a double-stranded polyribonucleotide and a polyalkyleneimine

ABSTRACT

The present invention relates to compositions comprising particles, each of said particles comprising a complex of at least one double-stranded polyribonucleotide, such as polyinosinic-polycytidylic acid [poly(I:C)], and at least one linear polyalkyleneimine. The particles are also characterized by their monomodal diameter distribution and z-average diameter within specific ranges. The present invention additionally relates to use of said compositions as medicaments, in particular for the treatment of a cell growth disorder characterized by abnormal growth of human or animal cells, as well as to processes for the preparation of said compositions.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticalformulations comprising particles formed by polyribonucleotides andpolymers, their production, and their medical use.

BACKGROUND TO THE INVENTION

The use of synthetic analogs of double-stranded RNA (dsRNA) that mimicviral dsRNA has been explored in recent years for specificallyactivating the immune system against tumors with the scope of inhibitingcancer cell growth and inducing cancer cell apoptosis. In particular,double-stranded polyinosinic-polycytidylic acid (named as poly(I:C) orpIC) has been characterized as a type of dsRNA with various effects oftherapeutic interest against various cancers (such as melanoma,hepatoma, colon, gastric, and oral carcinoma, cervical cancer, breastcancer, ovarian cancer, urinary tract tumors, lung and prostate cancer)and their metastasis, in manners that may be dependent or independentfrom immune system activation, natural killer- and/or dendriticcell-mediated activities, and/or changes of tumor gene expression andmicroenvironment (Hafner A et al., 2013).

Unfortunately, these initial preclinical evidences are poorly or notconfirmed in clinical studies with naked poly(I:C) molecules thatrevealed its low stability, poor homogeneity, unpredictablepharmacokinetics, and limited antitumoral effects due to a variety ofmechanisms, such as poor cellular uptake or degradation by cytosolicRNases (Hafner A et al., 2013). Indeed, in order to achieve an effectivetherapeutic or prophylactic effect, poly(I:C) molecules may need to bere-dissolved immediately prior or shortly before use, may be availablein formulations at low concentrations, and/or are frequentlyadministered (e.g. every 2 hours).

During the last few years, there has been significant progress informulating poly(I:C) molecules with immunomodulatory and/or therapeuticproperties. Various methods of preparing and formulating poly(I:C)molecules as powder and/or integrated within polymer-basedmicroparticles with or without targeting moieties and additionalchemical linkers have been disclosed (CN103599071; CN102988303;WO2004045491; WO2008057696; WO2013164380; WO2015067632, WO2014057432;WO2014165296; Schaffert D et al., 2011 ; WO2015173824, Kabilova T etal., 2014; Kübler K et al., 2011; Palchetti S et al., 2013; Saheki A etal., 2011). Poly(I:C) molecules have been formulated with carrierpolymers and in formats compatible for nasal administration(WO2013164380), stabilized with polylysine and carboxymethylcellulose(WO2005102278), encapsulated within cationic lipid-coated calciumphosphate nanoparticles, liposomes, or other vesicular structures (ChenL et al., 2013; US2009117306; US2011003883, or together with singlestranded RNA and with cationic peptides like protamine (WO2013087083).Alternatively, poly(I:C) molecules have also been immobilized on solidparticles and carriers such as iron oxide nanoparticles, with or withoutagents that would help targeting poly(I:C) molecules to specific cellsor tissues (McBain S et al., 2007; Cobaleda-Siles M et al., 2014).

Some publications further disclose various ternary or quaternarycomplexes in the sub-micrometer range that are formed by polymers,poly(I:C) and/or double stranded DNA, with or without other componentsand gene-specific (Kurosaki T et al., 2009.; WO2013040552,;WO2013063019,; Tutin-Moeavin I et al., 2015).

However, these approaches have the objective of providing agents thatessentially administer DNA to the cells, while maintaining theirviability, and not the selective killing of cancer cells.

The pitfalls that are limiting the clinical development of poly(I:C)molecules as a drug and its compliancy with regulatory requirementscould be overcome by producing structurally complex anticancer complexescomprising poly(I:C) molecules together with drug delivery systems forcancer therapy that are often based on cationic polymers such aschitosan, polyethyleneimine (PEI), poly-L-lysine, polymethacrylates,imidazole- or cyclodextrin-containing polymers, poly(beta-amino ester)s,and related dendrimers. These polymeric systems (also called asPolyplex) are structurally and functionally distinct from lipid-basedsystems (also called as Lipoplex) and hybrid systems (also called asLipopolyplex) that are similarly used for the local or systemicdelivering of nucleic acids (Bilensoy E, 2010; Germershaus O and NultschK, 2015). Among Polyplex, PEI is a cationic polymer of particularinterest that can be modified at the level of linear/branched structureand size, chemical linkage, degradability, and derivatization (Islam Met al., 2014) and that, differently from lipoplex internalization bycells, is internalized both by clathrin-mediated and by caveolaemediated endocytosis (Shabani M et al., 2010).

This therapeutic approach involving the preparation and theadministration of poly(I:C) molecules associated to PEI has beenexemplified in the literature by the agent called BO-110 (Pozuelo-RubioM et al., 2014; Tormo D et al., 2009; WO2011003883). This complex, alsoidentified as [pIC]^(PEI), not only engages a dual induction ofautophagy and apoptosis in several cancer cell lines of melanoma and ofother tumor types (such as gliomas or carcinomas) but also has no orlimited effect on the viability of normal cells, such as melanocytes.BO-110 inhibits melanoma growth in animal models for demonstratingantitumoral and antimetastatic activity in vivo, even in severelyimmunocompromised mice. Moreover, a similar [pIC]^(PEI)-based agentstimulates the apoptosis in pancreatic ductal adenocarcinoma cellswithout affecting normal pancreatic epithelial cells and in vivoadministration of [pIC]^(PEI) inhibited tumor growth in tumor animalmodels (Bhoopathi P et al., 2014). A further effect of BO-110administration is characterized in a model of endometriosis, whereinsuch agent reduces angiogenesis and cellular proliferation and increasesapoptosis (Garcia-Pascual C and Gomez R, 2013).

Thus, BO-110 and similar [pIC]^(PEI) agents that comprisedouble-stranded polyribonucleotides represent a novel anticancerstrategy with a broad spectrum of action, due to the combined activationof autophagy and apoptosis, autonomously and selectively in tumor cells,while maintaining the viability of normal cells of different lineages.However, BO-110, as for other double-stranded polyribonucleotide-basedagents that have demonstrated efficacy in various pre-clinical modelswhen associated with carriers, still needs to be provided informulations that are stable in different storage conditions, uniformlymanufactured and sized.

Indeed, prior art does not provide appropriate teaching for solvingissues related to the most effective combination of structural andbiophysical criteria that allow the production of poly(I:C)-containingcompositions for treatment of cancer. Regulatory agencies also requirebeing strictly compliant to the specifications on reproducibility,storage, and uniformity of the size and concentration ofpoly(I:C)-containing particles that are included within compositions foruse in humans. Thus, agents, compositions, and related processesproviding double-stranded polyribonucleotide molecules, such aspoly(I:C) molecules, at higher, and well-controlled, concentrations arestill needed to allow their extensive pre-clinical and clinicaldevelopment as a drug (in particular against cancer), while improvingpatient compliance and reducing the frequency of dosing double-strandedpolyribonucleotide molecules with well-defined safety margin andtherapeutic effects.

SUMMARY OF THE INVENTION

The present invention relates to a composition comprising particleswherein

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof;

(ii) at least 95%, or at least 90%, of said particles has a diameter ofless than or equal to 600 nm, preferably, less than or equal to 300 nm(for example, between 140 and 250 nm); and

(iii) said particles have a z-average diameter of less than or equal to200 nm, preferably less than or equal to 150 nm, in particular, asmeasured according to ISO 22412.

In a preferred embodiment, the present invention relates to an aqueouscomposition comprising particles wherein

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one linear polyalkyleneimine, or a salt and/or solvate thereof,wherein said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] and the average molecularweight of said linear polyalkyleneimine is between 17 and 23 kDa;

(ii) at least 90% of said particles has a mono-modal diameterdistribution below 300 nm;

(iii) said particles have a z-average diameter of less than or equal to150 nm, as measured according to ISO 22412; and

(iv) said composition has a zeta potential equal or superior to 30 mV,according to ISO 13099.

The present invention also relates to an aqueous composition comprisingparticles as disclosed herein wherein:

(i) each of said particles is formed by making a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one linear polyalkyleneimine, or a salt and/or solvate thereof,wherein said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] and the average molecularweight of said linear polyalkyleneimine is between 17 and 23 kDa;

(ii) at least 90% of said particles has a mono-modal diameter below 300nm;

(iii) said particles have a z-average diameter of less than or equal to150 nm, as measured according to ISO 22412; and

(iv) said composition has a zeta potential equal or superior to 30 mV,according to ISO 13099;

wherein said particles are formed at the ratio of the number of moles ofnitrogen of said polyalkyleneimine to the number of moles of phosphorusof said double-stranded polyribonucleotide in said composition beingequal to or greater than 2.5.

The present invention also relates to a composition obtainable bylyophilisation of the aqueous composition as disclosed herein.

In addition, the present invention relates to a composition, asdisclosed herein, for use as a medicament.

Moreover, the present invention relates to a composition, as disclosedherein, for use in treatment of a cell growth disorder characterized byabnormal growth of human or animal cells.

Furthermore, the present invention relates to a process to manufacturethe composition, as disclosed herein, which comprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 500 nm to form sterilizedsolutions;

(iii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate of greater than or equal to 1 mL/min, or by simultaneous additionof each of said solutions into said mixing chamber, optionally byinjection, at a rate of greater than or equal to 1 mL/min; andoptionally

(iv) filtering the resulting aqueous composition through a filter havinga pore diameter of less than or equal to 600 nm to form a filtrate, orcentrifuging the resulting aqueous composition at greater than or equalto 22480 m/s² to form a supernatant; and/or

(v) lyophilising the resulting aqueous composition, filtrate orsupernatant.

Preferably, the present invention also relates to a compositioncomprising particles wherein:

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof, wherein

-   -   (a) said double-stranded polyribonucleotide is        polyinosinic-polycytidylic acid [poly(I:C)] or        polyadenylic-polyuridylic acid [poly(A:U)], wherein at least 60%        of said double-stranded polyribonucleotides have at least 850        base pairs, at least 70% of said double-stranded        polyribonucleotides have between 400 and 5000 base pairs, and        between 20% and 45% of said double-stranded polyribonucleotides        have between 400 and 850 base pairs; and    -   (b) said polyalkyleneimine comprises at least 95%        polyethyleneimines, wherein the weight average molecular weight        of said polyalkyleneimine is between 17 and 23 kDa and the        polydispersity index is <1.5, and wherein the ratio of the        number of moles of nitrogen of said polyalkyleneimine to the        number of moles of phosphorus of said double-stranded        polyribonucleotide in said composition is between 2.5 and 5.5;

(ii) at least 99% of said particles has a diameter of less than or equalto 600 nm; and

(iii) said particles have a z-average diameter of between 30 nm and 150nm.

More preferably, the present invention also relates to an aqueouscomposition which comprises particles wherein:

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof, wherein

-   -   (a) said double-stranded polyribonucleotide is        polyinosinic-polycytidylic acid [poly(I:C)], wherein at least        60% of said poly(I:C) has at least 850 base pairs, at least 70%        of said poly(I:C) has between 400 and 5000 base pairs, and        between 20% and 45% of said poly(I:C) has between 400 and 850        base pairs; and    -   (b) said polyalkyleneimine is polyethyleneimine (PEI), wherein        the weight average molecular weight of said PEI is between 17.5        and 22.6 kDa and the polydispersity index is <1.5, and wherein        the ratio of the number of moles of nitrogen of said        polyalkyleneimine to the number of moles of phosphorus of said        double-stranded polyribonucleotide in said composition is        between 2.5 and 4.5;

(ii) at least 99% of said particles has a diameter of less than or equalto 500 nm;

(iii) said particles have a z-average diameter of between 60 nm and 130nm; and

(iv) said particles have a median diameter (D50%) of between 75 nm and150 nm.

Further embodiments related to the preparation of such compositions inform of BO-11X formulations, their features, their analysis and theiruses are provided in the Detailed Description and in the Examples below.

DESCRIPTION OF THE FIGURES

FIG. 1: Functional activity of distinct BO-111 preparations followingfiltration or centrifugation by analyzing cell death in melanoma cellsSK-Mel-103 after a 48-hour exposure. (A) Poly(I:C) molecules areassociated with PEI to form BO-111 complexes according to thedescription of the 2-vial manufacturing processing in Example 1, and theresulting solution is either filtered using membrane with different poresizes (and then using the flow-through solution for cell-based assays)or centrifuged at different speeds (and then using the supernatant forcell-based assays), generating distinct BO-111 preparations, each ofthem containing poly(I:C)-based complexes with a more homogeneous, lowersize that is determined as Z-average (hydrodynamic diameter, expressedas d·nm). Qualitatively similar data were generated by testing celldeath at 24 hours. Data on cell death are compared with sample nottreated with BO-111. (B) Dose-response activity of a BO-111 preparationthat is obtained by filtering through 0.8 μm filter (hatched bars) whencompared to the corresponding unfiltered BO-111 preparation (whitebars). Since centrifugation or filtration causes some loss of poly(I:C)due to the sedimentation or retention of bigger complexes(respectively), the amount of poly(I:C) in each distinct BO-111preparation was quantified by spectrophotometric analysis at wavelengthof 260 nm, in order to compare correctly the effects of such preparationon cells by diluting accordingly the initial preparation at theappropriate concentration. Qualitatively similar data were generated bytesting cell death at 12, 24, and 36 hours. Cell death is determined byTrypan Blue assay as described in the literature. Standard deviation iscalculated using triplicate data for each condition of the sameexperiment.

FIG. 2: Effects of PEI features on BO-111 functional activity, asdetermined using the same cell-based assay and BO-111 concentration ofFIG. 1. (A) Distinct BO-111 preparations were obtained by usingcommercial JetPEI preparations containing linear polymers havingdifferent molecular weight (expressed in kiloDalton; NT: control, nottreated cells; such JetPEI preparations present a mono-modaldistribution with polydispersity index that is inferior to 1.5, inparticular comprised between 1.1 and 1.4). (B) Distinct BO-111preparations were obtained by modifying the ratio N/P (from 1.2 to 5.3)that is calculated as: μL PEI×150 nM/μg poly(I:C)×3 nmol, wherein 150 nMrefers to the concentration of nitrogen residues in PEI and 3 nmolrefers to the number of nanomoles of anionic phosphate per 1 μgpoly(I:C). The pharmacological effect of each combination is determinedby comparing cell death percentages in both melanoma cells andmelanocytes (control cells; NT: control, not treated cells). The samepoly(I:C) preparation as described in FIG. 1 was used for all BO-111preparations that are described in (A) and (B) but, instead of applyingthe “fast pipetting” method, poly(I:C) solution was quickly injected inthe vial containing the PEI solution using a syringe, increasing themixing speed (at least as it can be visually determined). Standarddeviation is calculated using triplicate data from each condition of thesame experiment.

FIG. 3: Analysis of poly(I:C) molecules within BO-111 preparations byelectrophoresis. (A) Unlabelled preparations of initial poly(I) orpoly(I:C) molecules are compared to increasing amounts of BO-111preparations in agarose gel 0.8% (1 hour electrophoresis). The molecularsize of poly(I:C) molecules is determined in comparison with sizemarkers. (B) Radiolabeled poly(I:C) molecules are compared to unlabeledpoly(I:C) and to a BO-111 preparation including the same radiolabeledpoly(I:C), using an agarose gel 1% (1-hour electrophoresis) that isexposed to a photographic film. The poly(I:C) molecules within thedifferent BO-111 preparations that are not visible in panel (A) appearin panel (B) as being blocked within the well of agarose gel since theyare associated to JetPEI forming particles that (due to their sizeand/or charge distribution) the electric field cannot displace acrossthe agarose gel. (C) Unlabelled poly(I:C) molecules are compared withdistinct BO-111 preparations obtained by modifying the ratio N/P (1.2, 3and 5.3; this value is calculated as in FIG. 2) in agarose gel 0.8%(1-hour electrophoresis). Each sample is either untreated (C+) ortreated with a specifically degrading enzyme (RNAse; Rnase A: 5 μg/mLover 30 minutes) prior to be loaded on the agarose gel, in order toevaluate the stability of poly(I:C) molecules in different BO-111preparations. The poly(I:C) molecules appear insensitive to such enzymewhen complexed with JetPEI at higher N/P ratios (greater than or equalto 3), and a smear of poly(I:C) molecules appears released when the N/Pratio is below 3. In panels (A) and (C), the molecular size of poly(I:C)molecules is determined in comparison with size marker.

FIG. 4: Size distribution of BO-111 complexes in preparations that areobtained using the 2-vial production process, as determined by comparingsignal intensity using Dynamic Light Scattering by zeta sizer nano ZStechnology. (A) Comparison of particle diameter distribution wasperformed using three different BO-111 batches obtained by using thesame production process. (B) Comparison of distinct BO-111 preparationswhen exposing (or not, sample indicated as CONTROL) to an incubation atroom temperature in agitation for 30 minutes. (C) Comparison of distinctBO-111 preparations when exposing (or not, sample indicated as CONTROL)to an incubation at 50° C. for a period of 30 minutes. (D) Comparison ofdistinct BO-111 preparations, control manufacture as suggested inExample 1 and “Slow Mixing” sample, evaluated by triplicate profiles,that was obtained by the drop-by-drop approach using a reduced mixingspeed. Particle size is evaluated considering particle diameter innanometers (d·nm).

FIG. 5: Scheme of GMP manufacturing of BO-11X compounds, as exemplifiedby BO-112. (A) Flowchart summarizing main steps for manufacturing BO-112compounds as a GMP-compliant, pharmaceutical preparation comprisingpoly(I:C) molecules, commercial JetPEI preparations, and glucose. (B)Permeation Chromatograms for BO-112, JetPEI, and glucose samples thatare analyzed using refractive index detection (RI). The BO-112 signaloverlaps with the glucose signal, while the characteristic peak of freeJetPEI at the Retention Volume of 13.75 mL disappeared, suggesting thatall initial JetPEI was incorporated into BO-112. (C) Modification ofBO-112 compound distribution due to increased speed of mixing duringmanufacture process at low (below 100 rpm) or at high (beyond 100 rpm,up to 550 rpm) injection speed, where 550 rpm is the reference speed formixing solutions in the BO-112 manufacturing process, that is forobtaining particles with Z-average diameter (d· nm) going from around100 nm down to 73 nm, 54 nm or less (functional BO-112 preparationspresent an average diameter between 30 nm and 150 nm; particle diameteris defined as in previous figures in d· nm).

FIG. 6: Structural analysis of different poly(I:C) formulations. (A) Themolecular size of different poly(I:C) preparations is determined inagarose gel using size markers. (B) Size distribution of BO-112complexes is compared to three poly(I:C)-containing commercial products(Poly-ICLC, LyoVec-HMW, and LyoVec-LMW; particle size is defined as inprevious figures in nm).

FIG. 7: Changes in the size distribution following storage at −20° C. isdetermined for BO-112 (A), Poly-ICLC (B), LyoVec-HMW (C), and LyoVec-LMW(D), as determined by comparing signal intensity using Nanosizertechnology (particle diameter is defined as in previous figures in nm).

FIG. 8: Effect of different poly(I:C) formulations on cell viability intwo distinct cancer cell models. BO-112 is compared to untreated cellsand to cells treated with Poly-ICLC, LyoVec-LMW, or LyoVec-HMW, eachformulation being tested at the indicated concentrations that weredetermined according to complex weight but with a similar content ofpoly(I:C) molecules in either a melanoma (A) or pancreatic cancer (B)cell model. The cell viability data were generated using crystal violetassay method after 24 hours. (C) Effect of different poly(I:C)formulations on signaling molecules of therapeutic interest.Interferon-beta (IFN-beta) expression was evaluated by RT-qPCR method inSK-MEL-103 cells that were exposed to BO-112, Poly-ICLC (or untreated,NT) for 8, 16 and 24 hours. BO-112 and Poly-ICLC formulations were usedat a concentration providing a similar content of poly(I:C) molecules.

FIG. 9: Effect of linear PEI alone, poly(I:C), and two differentPEI/poly(I:C) formulations (BO-110 and BO-112;) on the viability ofnormal, primary melanocytes (preparations #1 and #2) and four cancercell lines, demonstrating the specific cytotoxicity of suchPEI/poly(I:C) formulations against cancer cell lines. Poly(I:C) contentused in preparation of the Poly(I:C) only, BO-110, and BO-112 treatmentsadministered is identical (1 μg/mL per 40 hours of treatment).

FIG. 10: Effect on BO-112 administration in an animal model for humancancer. (A) Timeline showing the schedule of treatment. All mice wereinjected sub-cutaneously with B16-F10 murine melanoma cells at day 0.After randomizing mice into five groups presenting tumors of similaraverage size (80-100 mm³) on day 7, animals were treated with BO-112formulation (double circles) by injection straight into the tumor tissue(i.t. treatment) at 3 different concentrations in groups 2, 3, and 4(G2, G3, G4) or, in the two remaining groups (G1 and G5), with vehicleonly. On the following treatment days (10, 14, 17, 21 and 24) all groupsbut G1, received intraperitoneal (i.p.) administration of an anti-PD-L1murine antibody (150 μg/dose) in addition to the intratumoraladministration of BO-112 formulation at the same concentration of day 7.Survival was monitored daily, and mice were scored as dead upon findingthem deceased, or when the tumor volume reached the maximum allowedsize. Monitoring continued after last treatment until day 45, when thelast mouse died and the experiment was terminated. (B) Survival curvecomparing the control groups G1 and G5 with the three groups in whichBO-112 formulation was administered at the indicated threeconcentrations. When comparing the groups, there was a statisticaldifference (p<0.0001, Log-rank Mantel-Cox test) between the controlgroups and the test groups, with G4 showing the strongest increase ofsurvival relative to vehicle or anti-PD-L1 alone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition comprising particleswherein:

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof;

(ii) at least 95% of said particles has a diameter of less than or equalto 600 nm, preferably, less than or equal to 300 nm; and

(iii) said particles have a z-average diameter of less than or equal to200 nm, preferably less than or equal to 150 nm, in particular, asmeasured according to ISO 22412.

In a preferred embodiment, the present invention relates to an aqueouscomposition comprising particles wherein

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one linear polyalkyleneimine, or a salt and/or solvate thereof,wherein said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] and the average molecularweight of said linear polyalkyleneimine is between 17 and 23 kDa;

(ii) at least 90% of said particles has a mono-modal diameterdistribution below 300 nm;

(iii) said particles have a z-average diameter of less than or equal to150 nm, as measured according to ISO 22412; and

(iv) said composition has a zeta potential equal or superior to 30 mV,according to ISO 13099

The present invention also relates to an aqueous composition comprisingparticles as disclosed herein wherein:

(i) each of said particles is formed by making a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one linear polyalkyleneimine, or a salt and/or solvate thereof,wherein said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] and the average molecularweight of said linear polyalkyleneimine is between 17 and 23 kDa;

(ii) at least 90% of said particles has a mono-modal diameterdistribution below 300 nm;

(iii) said particles have a z-average diameter of less than or equal to150 nm, as measured according to ISO 22412; and

(iv) said composition has a zeta potential equal or superior to 30 mV,according to ISO 13099;

wherein said particles are formed at the ratio of the number of moles ofnitrogen of said polyalkyleneimine to the number of moles of phosphorusof said double-stranded polyribonucleotide in said composition beingequal to or greater than 2.5.

The particles that are made of and formed by said complexes may presentadditional features, as per the disclosure below, such that in furtherembodiments said particles may comprise further components such asexcipients like mannitol or glucose, or the absence of further elements,such as cancer-targeting functionality or other moieties and linkers.Additional features can be defined in further preferred embodiments whenthe particles are provided and analysed within the compositions [i.e.within the liquid (aqueous) or lyophilised formulations], such as whendefined as having a mono-modal size distribution within specific ranges,for example, between 30 nm and 150 nm, or when the composition ischaracterised by the absence of single-stranded polyribonucleotidemolecules (as established by a low or absent hyperchromic effect). Otherfeatures as defined in accordance to internationally establishedstandards that are required for regulatory approval and/or GoodManufacturing Processes are disclosed in the following.

In a preferred embodiment of the composition of the present invention,at least 40% of the double-stranded polyribonucleotides comprised insaid composition have at least 850 base pairs, and at least 50% of thedouble-stranded polyribonucleotides comprised in said particles havebetween 400 and 5000 base pairs. Moreover, the double-strandedpolyribonucleotides that are comprised in the complexes may presentspecific ranges of lengths that are defined by their processes ofpreparation and/or according to the desired use. For example, at least40%, 50%, 60%, or any other higher percentage of the double-strandedpolyribonucleotides comprised in said particles may have at least 850base pairs, and at least 50%, 60%, 70% or any other higher percentage ofthe double-stranded polyribonucleotides comprised in said particles mayhave between 400 and 5000 base pairs. Additional ranges that may definedouble-stranded polyribonucleotides that are comprised in said particlesare:

(i) between 5% and 60% (and preferably between 10% and 30%) ofdouble-stranded polyribonucleotides having less than 400 base pairs;

(ii) between 20% and 45% (or between 15% and 30%, but preferably between20% and 30%) of double-stranded polyribonucleotides having between 400and 850 base pairs;

(iii) between 20% and 70% (and preferably between 50% and 60%) ofdouble-stranded polyribonucleotides having between 850 and 5000 basepairs; and/or

(iv) between 0% and 10% (and preferably 1% or less) of double-strandedpolyribonucleotides having more than 5000 base pairs.

Thus, in more preferred embodiment of the composition of the presentinvention, at least 50% of the double-stranded polyribonucleotidescomprised in said composition have at least 850 base pairs, and at least60% of the double-stranded polyribonucleotides comprised in saidcomposition have between 400 and 5000 base pairs. Yet more preferably,at least 60% of the double-stranded polyribonucleotides comprised insaid composition have at least 850 base pairs, at least 70% of saiddouble-stranded polyribonucleotides comprised in said composition havebetween 400 and 5000 base pairs, and between 20% and 45% of saiddouble-stranded polyribonucleotides have between 400 and 850 base pairs.Even more preferably, at least 60% of the double-strandedpolyribonucleotides comprised in said composition have at least 850 basepairs, at least 70% of said double-stranded polyribonucleotidescomprised in said composition have between 400 and 5000 base pairs,between 20% and 30% of said double-stranded polyribonucleotides havebetween 400 and 850 base pairs, and between 10% and 30% of saiddouble-stranded polyribonucleotides have less than 400 base pairs.

In one embodiment of the present invention, the double-strandedpolyribonucleotide is preferably polyinosinic-polycytidylic acid[poly(I:C)] molecules or polyadenylic-polyuridylic acid [poly(A:U)]molecules. More preferably, the double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] molecules. Saiddouble-stranded polyribonucleotide molecules comprise strands of, forexample, poly(I) and poly(A) that pair with poly(C) and poly (U),respectively, thus forming double-stranded polyribonucleotides, whereineach strand may comprise up to 5% of ribonucleotides different from themajority of ribonucleotides in said strand and/or comprise up to 5%mismatched base pairs, more preferably up to 1% of ribonucleotidesdifferent from the majority of ribonucleotides in said strand, and/orcomprise up to 1% mismatched base pairs. Depending on the selectedpolyribonucleotide and/or the process for generating said complexes, afraction of the polyribonucleotides comprised in the complex may alsocomprise single-stranded (i.e. non-paired) polyribonucleotides.

In a preferred embodiment, the content of free, single-strandedpolyribonucleotide within these particles and the compositions isevaluated on the basis of the hyperchromicity (or hyperchromic effect).This effect is due to the increase of optical density (absorbance) ofdouble stranded polynucleotides when this duplex structure is denatured.The UV (ultraviolet) absorption of polynucleotides is increased when thetwo single strands are being separated, either by heat or by addition ofdenaturant or by increasing the pH level. Hyperchromicity can thereforebe used to check the structures of poly(I:C) or poly(A:U) moleculeswithin the particles as temperature (or another condition) changes,thereby respectively determining the separation between poly(I) strandsand poly(C) strands in poly(I:C) molecules or the separation betweenpoly(A) strands and poly(U) strands in poly(A:U) molecules. Preferably,such content of single-stranded poly(I) molecules and poly(C) moleculesor single-stranded poly(A) molecules and poly(U) molecules in theparticles is as low as possible, as determined by measuring absorbanceof light in the 260 nm wavelength region using standard equipment andprotocols. For instance, this effect can be measured using aspectrophotometer and according to European Pharmacopoeia (2.2.25;Absorption spectrophotometry, ultraviolet and visible). In particular,the compositions disclosed herein exhibit less than a 20% increase inabsorption at 260 nm between 20° C. and 80° C., preferably less than a10%, more preferably less than a 5%, even more preferably, a less than1% increase in absorption at 260 nm between 20° C. and 80° C.Alternatively, the compositions disclosed herein show a less than 0.2increase in the transmittance between room temperature and 40° C., 50°C., 60° C., 70° C., 80° C. and 90° C. This value can be measured as theabsorbance at 260 nm (A) or the transmittance (1/A) at room temperatureand then calculating the difference with the respective value ofabsorbance or transmittance at the aforementioned higher temperatures.

In a preferred embodiment of the composition of the present invention,the polyalkyleneimine, or a salt and/or solvate thereof, is linear,branched and/or dendritic, more preferably said polyalkyleneimine is alinear polyalkyleneimine. In a more preferred embodiment, saidpolyalkyleneimineis a homo-polyalkyleneimine orhetero-polyalkyleneimine. The polycationic homo- or hetero-polymerpreferably comprises a repeating unit formed by an amine group and atleast a two carbon atom spacer, thus comprising homo-polyalkyleneimineor hetero-polyalkyleneimine polymers which are linear or branched and/ordendritic. Examples of polyalkyleneimines are polyethyleneimine,polypropyleneimine, polybutyleneimine and polypentyleneimine, mixedpolymers of any of these homopolymers, or any commercially available orotherwise disclosed derivatives. This polyalkyleneimine polymer ispreferably water-soluble. In a particularly preferred embodiment of thepresent invention, said polyalkyleneimine is a water-soluble, linearhomo-polyalkyleneimine or hetero-polyalkyleneimine. In an even morepreferred embodiment, said polyalkyleneimine comprises in particular awater-soluble linear homo-polyalkyleneimine, more preferably itcomprises at least 75% linear polyethyleneimines, yet more preferably95% linear polyethyleneimines. In a still more preferred embodiment ofthe composition of the present invention said polyalkyleneimine ispolyethyleneimine (also known as PEI). Thus, in an especially preferredembodiment, said polyalkyleneimine is a linear polyethyleneimine.

In another preferred embodiment of the composition of the presentinvention, the weight average molecular weight of said polyalkyleneimineis between 17 and 23 kDa, still more preferably between 17.5 and 22.6kDa, and has a molecular weight polydispersity index of <1.5. Saidweight average molecular weight and said polydispersity index weredetermined for the polyalkyleneoxide precursor to said polyalkyleneimineaccording to ISO 16014:2012, preferably by Gel Permeation Chromatography(GPC) according to ISO 16014-2:2012. The polydispersity index iscalculated as M_(w)/M_(n) (weight average molecular weight/numberaverage molecular weight) and is inferior to 1.5.

In another preferred embodiment of the composition of the presentinvention, the ratio of the number of moles of nitrogen of saidpolyalkyleneimine to the number of moles of phosphorus of saiddouble-stranded polyribonucleotide in said composition is equal to orgreater than 2.5, more preferably between 2.5 and 5.5, still morepreferably between 2.5 and 4.5, furthermore preferably between 2.5 and3.5. This ratio is particularly important when forming the particleswithin the composition and providing compositions having the desiredeffects and properties.

Thus, as mentioned above, the present invention relates to an aqueouscomposition comprising particles as disclosed herein wherein:

(i) each of said particles is formed by making a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one linear polyalkyleneimine, or a salt and/or solvate thereof,wherein said double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] and the average molecularweight of said linear polyalkyleneimine is between 17 and 23 kDa;

(ii) at least 90% of said particles has a mono-modal diameterdistribution below 300 nm;

(iii) said particles have a z-average diameter of less than or equal to150 nm, as measured according to ISO 22412; and

(iv) said composition has a zeta potential equal or superior to 30 mV,according to ISO 13099;

wherein said particles are formed at the ratio of the number of moles ofnitrogen of said polyalkyleneimine to the number of moles of phosphorusof said double-stranded polyribonucleotide in said composition beingequal to or greater than 2.5, more preferably between 2.5 and 5.5, stillmore preferably between 2.5 and 4.5, furthermore preferably between 2.5and 3.5. In a further preferred embodiment, said composition is formedby making a complex from at least 0.5 mg of polyinosinic-polycytidylicacid [poly(I:C)] per mL of the total (i.e. final) volume of saidcomposition, more preferably from at least 2.0 mg of poly(I:C) per mL ofthe total volume of said composition. Thus, in a particularly preferredembodiment of the invention, the double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)] or polyadenylic-polyuridylicacid [poly(A:U)], wherein at least 60% of said double-strandedpolyribonucleotides have at least 850 base pairs (bp), at least 70% ofsaid double-stranded polyribonucleotides have between 400 and 5000 basepairs, and between 20% and 45% of said double-strandedpolyribonucleotides have between 400 and 850 base pairs; and thepolyalkyleneimine comprises at least 95% polyethyleneimines, wherein theweight average molecular weight of said polyalkyleneimine is between 17and 23 kDa and the polydispersity index is <1.5, and wherein the ratioof the number of moles of nitrogen of said polyalkyleneimine to thenumber of moles of phosphorus of said double-stranded polyribonucleotideused in formation of said composition (i.e. the ratio of the number ofmoles of nitrogen of said polyalkyleneimine to the number of moles ofphosphorus of said double-stranded polyribonucleotide used in formationof said particles) is between 2.5 and 5.5.

In an even more preferable particularly preferred embodiment of theinvention, the double-stranded polyribonucleotide ispolyinosinic-polycytidylic acid [poly(I:C)], wherein at least 60% of thepoly(I:C) molecules have at least 850 base pairs, at least 70% of saidpoly(I:C) molecules have between 400 and 5000 base pairs, between 20%and 30% of said poly(I:C) molecules have between 400 and 850 base pairs,and between 10% and 30% of said poly(I:C) molecules have less than 400base pairs; and the polyalkyleneimine is polyethyleneimine, wherein theweight average molecular weight of said polyalkyleneimine is between17.5 and 22.6 kDa and the polydispersity index is <1.5 (such as between0.1 and 0.6, as measured within the composition), and wherein the ratioof the number of moles of nitrogen of said polyethyleneimine to thenumber of moles of phosphorus of said poly(I:C) used in formation ofsaid composition (i.e. the ratio of the number of moles of nitrogen ofsaid polyalkyleneimine to the number of moles of phosphorus of saiddouble-stranded polyribonucleotide used in formation of said particles)is between 2.5 and 4.5.

In the present invention, the z-average diameter and polydispersityindex of the diameters of the particles comprised in the composition ofthe present invention are determined by Dynamic Light Scattering (DLS)techniques, based on the assumption that said particles are isotropicand spherically shaped. In particular, the z-average diameter(zeta-average diameter) refers to the intensity-weighted arithmeticaverage hydrodynamic diameter of said particles, as determined accordingto industrial standard ISO 22412:2008. In addition, industrial standardISO 22412:2008 provides a measure of the particle diameter (size)distribution in the form of a polydispersity index and allowscalculation of the percentiles (D values) known as D50% (the maximumparticle diameter below which 50% of sample intensity falls, also knownas the median diameter), D90% (the maximum particle diameter below which90% of sample intensity falls), D95% (the maximum particle diameterbelow which 95% of sample intensity falls), and D99% (the maximumparticle diameter below which 99% of sample intensity falls). Thus,based on the methodology presented in ISO 22412, it is possible toascertain that at least 95% (D95) or preferably 90% (D90) of particlescomprised in the composition of the present invention has a diameter ofless than or equal to 600 nm, more preferably less than or equal to 300nm, and that said particles have a z-average diameter of less than orequal to 200 nm, more preferably less than 150 nm.

In a preferred embodiment of the composition of the present invention,said particles have a mono-modal diameter distribution, in particularwithin the sub-micrometer range indicated above. Indeed, in one aspectthe aqueous composition of the present invention comprises particleswherein at least 90% of said particles has a mono-modal diameterdistribution below 300 nm, wherein said particles have a z-averagediameter of less than or equal to 150 nm, as measured according to ISO22412. Particles (or their aggregates) having a size superior to suchvalues (e.g. in the micrometer range, such as above 10 μm) that may bestill present (but, in any case below the limits indicated in EuropeanPharmacopoeia) can be removed by filtration, at the end of manufacturingand/or just before administration (for example, through 0.8 micrometerfilter). Thus, all or the large majority of particles comprised in thiscomposition may present a mono-modal diameter distribution within thecomposition that, as shown in the Examples, is established during theirpreparation and can be maintained and adapted according to the desireduse and/or storage.

In another preferred embodiment of the present invention at least 95% or90% of said particles has a diameter of less than or equal to 600 nm(i.e. the maximum particle diameter below which 95% or 90% of sampleintensity falls=D95% or D90%=600 nm), more preferably not exceeding thediameter of 500 nm, still more preferably not exceeding the diameter of400 nm, and yet more preferably not exceeding the diameter of 300 nm.Within such limits, Even more preferably, at least 99% of said particleshas a diameter of less than or equal to 600 nm, yet more preferably atleast 99% of said particles has a diameter of less than or equal to 500nm, much more preferably at least 99% of said particles has a diameterof less than or equal to 400 nm and yet more preferably not exceedingthe diameter of 300 nm. On the other hand, in a preferred embodiment,said particles have a median diameter (D50%) between 75 and 150 nm, morepreferably between 80 and 130 nm, and a D90% of between 140 and 250 nm,more preferably between 170 and 240 nm.

In another preferred embodiment of the present invention, said particleshave a z-average diameter below 150 nm, and more preferably in rangescomprised between 30 nm and 150 nm (such as furthermore preferablybetween 50 nm and 150 nm, between 75 nm and 150 nm, between 50 nm and100 nm, between 100 nm and 150 nm, or between 60 nm and 130 nm). Morepreferably, said particles of the aqueous composition of the presentinvention have a mono-modal diameter distribution between 30 nm and 150nm.

Thus, in most particularly preferred embodiments: (i) at least 99% ofparticles comprised in the composition of the present invention have adiameter of less than or equal to 600 nm, whereby said particles have az-average diameter of between 30 nm and 150 nm; and (ii) at least 99% ofparticles comprised in the composition of the present invention have adiameter of less than or equal to 500 nm, whereby said particles have az-average diameter of between 60 nm and 130 nm and have a mediandiameter (D50%) between 75 and 150 nm.

In a preferred embodiment of the present invention, said composition isobtainable by lyophilisation of the aqueous compositions disclosedherein. Thus, the composition of the invention may be an aqueous or alyophilised composition. Thus, the composition of the present invention(hereinafter BO-11X formulation, where X may be a whole number such thata BO-11X formulation encompasses, for example, a BO-111 and a BO-112formulation) can be provided in a solid (as a lyophilized or otherhighly concentrated form of the particles), semi-solid (as a gel), orliquid form, but is preferable as a liquid composition (such as anaqueous composition or other type of particle suspension that can beinjected or inhaled). The BO-11X formulations can be used, shipped, andstored as such, or can be used for obtaining a lyophilized form forspecific uses, shipment, storage, administration with other compounds,and/or further technical requirements. With respect to thelyophilisation process and equipment, classical freeze-drying or morerecent methods (such as electro-freezing, ultrasound-controlled or icefog), may be adapted for handling and manufacturing of the BO-11Xformulations, also by changing parameters such as pH, drying air speed,time, humidity, pressure, or temperature.

Thus, in one most preferred embodiment, the composition of the presentinvention comprises particles wherein:

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof, wherein

-   -   (a) said double-stranded polyribonucleotide is        polyinosinic-polycytidylic acid [poly(I:C)] or        polyadenylic-polyuridylic acid [poly(A:U)], wherein at least 60%        of said double-stranded polyribonucleotides have at least 850        base pairs, at least 70% of said double-stranded        polyribonucleotides have between 400 and 5000 base pairs, and        between 20% and 45% of said double-stranded polyribonucleotides        have between 400 and 850 base pairs; and    -   (b) said polyalkyleneimine comprises at least 95%        polyethyleneimines, wherein the average molecular weight of said        polyalkyleneimine is between 17 and 23 kDa and the        polydispersity index is <1.5, and wherein the ratio of the        number of moles of nitrogen of said polyalkyleneimine to the        number of moles of phosphorus of said double-stranded        polyribonucleotide used in formation of said composition (i.e.        the ratio of the number of moles of nitrogen of said        polyalkyleneimine to the number of moles of phosphorus of said        double-stranded polyribonucleotide used in formation of said        particles) is between 2.5 and 5.5;

(ii) at least 99% of said particles has a diameter of less than or equalto 600 nm, preferably at least 95% of said particles has a diameter ofless than or equal to 400 nm; and

(iii) said particles have a z-average diameter of between 30 nm and 150nm.

In another most preferred embodiment, the composition of the presentinvention is an aqueous composition which comprises particles wherein:

(i) each of said particles comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof, wherein

-   -   (a) said double-stranded polyribonucleotide is        polyinosinic-polycytidylic acid [poly(I:C)], wherein at least        60% of said poly(I:C) has at least 850 base pairs, at least 70%        of said poly(I:C) has between 400 and 5000 base pairs, and        between 20% and 45% of said poly(I:C) has between 400 and 850        base pairs; and    -   (b) said polyalkyleneimine is polyethyleneimine (PEI), wherein        the weight average molecular weight of said PEI is between 17.5        and 22.6 kDa and the polydispersity index is <1.5, and wherein        the ratio of the number of moles of nitrogen of said        polyalkyleneimine to the number of moles of phosphorus of said        double-stranded polyribonucleotide used in formation of said        composition (i.e. the ratio of the number of moles of nitrogen        of said polyalkyleneimine to the number of moles of phosphorus        of said double-stranded polyribonucleotide used in formation of        said particles) is between 2.5 and 4.5;

(ii) at least 99% of said particles has a diameter of less than or equalto 500 nm, preferably at least 95% or 90% of said particles has amono-modal diameter distribution below 300 nm;

(iii) said particles have a z-average diameter of between 60 nm and 130nm; and

(iv) said particles have a median diameter (D50%) between 75 and 150 nm.

The BO-11X formulations can, in a preferred embodiment of the presentinvention, be provided as compositions further comprising apharmaceutically acceptable carrier, excipient, organic solvent, and/oradjuvant [such as glycerol, ethanol, glucose or mannitol, preferablyglucose or mannitol, more preferably in a concentration of between 1 and10% (weight/volume)] [i.e. wherein said composition is formed byadditionally adding glucose or mannitol in a concentration of between 1and 10% (weight/total volume of said composition)] that is best adaptedto the preferred final form (such as liquid or lyophilised), uses,shipment, storage, administration with other compounds, and/or furthertechnical requirements. In a more preferred embodiment, said compositionfurther comprises at least one compound selected from an organiccompound, an inorganic compound, a nucleic acid, an aptamer, a peptideor a protein.

In one aspect, the aqueous composition of the present invention has azeta potential equal or superior to 30 mV, preferably between 35 and 50mV or between 38 and 45 mV, still more preferably between 40 and 45 mV,according to ISO 13099.

In another embodiment of the composition of the present invention, saidcomposition is an aqueous composition that has:

(i) a pH of between 2 and 4;

(ii) an osmolarity of between 200 and 600 mOsm/kg;

(iii) a specific optical rotation of between +1500 and +3750degrees.mL·g⁻¹·dm⁻¹ at a wavelength of 589 nm at 20° C. when saidaqueous composition comprises 5% (weight/volume) D-glucose in water,referenced against water; and/or

(iv) a zeta potential greater than or equal to 30 mV (for instance,comprised between 35 and 50 mV).

In an even more preferred embodiment of the present invention, saidcomposition is an aqueous composition that has:

(i) a pH of between 2 and 4;

(ii) an osmolarity of between 200 and 600 mOsm/kg; and/or

(iii) a zeta potential greater than or equal to 30 mV (for instance,comprised between 35 and 50 mV).

In one especially preferable embodiment said composition is an aqueouscomposition comprising glucose or mannitol that has:

(i) a pH of between 2 and 4; and/or

(ii) an osmolarity of between 200 and 600 mOsm/kg.

In one, furthermore preferable embodiment of the present invention, saidcomposition is an aqueous composition comprising glucose that has:

(i) a pH of between 2.7 and 3.4;

(ii) an osmolarity of between 200 and 340 mOsm/kg; and/or

(iii) a zeta potential comprised between 35 and 50 mV or between 38 and45 mV, still more preferably between 40 and 45 mV.

In an alternative, furthermore preferable embodiment said composition isan aqueous composition comprising mannitol that has:

(i) a pH of between 2.7 and 3.4;

(ii) an osmolarity of between 200 and 600 mOsm/kg; and/or

(iii) a zeta potential comprised between 35 and 50 mV or between 38 and45 mV, still more preferably between 40 and 45 mV.

For the purposes of the present invention, the osmolarity valuesreported herein are strictly olmolality values, but since the density ofwater (the solvent in which the aqueous compositions of the presentinvention are made) approximates to 1.00 g/mL at 20° C. these terms areused interchangeably. For the purposes of the present invention, roomtemperature refers to a temperature between 20 and 25° C.

In the present invention, the zeta potential is measured according toISO 13099, preferably 13099-2:2012.

The present invention also relates to a composition, as disclosedherein, for use as a medicament. Analogously, the present inventiontherefore also relates to use of the composition, as disclosed herein,for the manufacture of a medicament. This use may be achieved byproviding the composition as liquid (aqueous) or lyophilised compositionwherein poly(I:C) molecules and the particles comprising it are in ahighly stable form and at high concentration {e.g, wherein saidcomposition is formed by making a complex using at least 0.5 mg, 0.7mg/mL, 0.9 mg/ml, up to 2.0 mg of polyinosinic-polycytidylic acid[poly(I:C)] or polyadenylic-polyuridylic acid [poly(A:U)] or more per mLof the total (i.e. final) volume of said composition.

These compositions are particularly adapted for direct administration tothe cancer cells, for example by means of intratumoral or peritumoralinjection into skin or an internal organ or tissue comprising suchtumors and cancer cells. In a preferred embodiment of the presentinvention, said medicament is injectable. In a more preferred embodimentof the present invention, said medicament is an injectable, aqueouscomposition, optionally comprising a pharmaceutically acceptablecarrier, excipient and/or adjuvant. This injectable, aqueous compositioncan be provided as such or after diluting a concentrated preparation ofpoly(I:C) or poly(A:U) molecules (at a respective concentration of atleast 0.5 mg of poly(I:C) or poly(A:U)/mL of the total volume ofcomposition to be made, or more, as established when preparing theparticles in terms of the respective weight of poly(I:C) or poly(A:U)molecules that are added to a given volume of solution) or a lyophilisedcomposition in order to make up a total volume of the composition of theinvention. This means that said composition is provided in the foregoingconcentrations determined in terms of the weight of poly(I:C) orpoly(A:U) employed in making the complex per volume of the total aqueouscomposition, but may be concentrated where appropriate, especially forlong-term storage and/or intratumoral administration. In particular, theBO-11X formulation with double-stranded poly(I:C) molecules at such highconcentrations (i.e. that made from particles comprising a complexformed by complexing at least 0.5 mg up to 0.7 mg, preferably 0.9 mg,more preferably 2.0 mg or more, of poly(I:C) with linear PEI per mL ofthe total aqueous composition) is most appropriate for administrationand use as a medicament. The intratumoral or peritumoral injection ofsuch a composition (depending also on the actual accessibility and/orsize of the tumor mass as evaluated by the practitioner) in one or moresmall or restricted locations where tumors and cancer cells are present,may provide a stronger and/or more timely therapeutic effect.

Moreover, the present invention additionally relates to a composition,as disclosed herein, for use in treatment of a cell growth disordercharacterized by abnormal growth of human or animal cells. Suchtreatment may involve the combined administration of the composition ofthe present invention with another therapeutic treatment such as anothercancer-specific drug (being an antibody targeting a cancer antigen or acancer vaccine, for example) or a standard-of-care treatment (such asradio- or chemotherapy). The combined administration may provide notonly an additive therapeutic effect but also other valuable effects suchas reduce the dosage of the other drug necessary to achieve a similartherapeutic benefit, reduce side effects, or provide a longer-lastingand/or a synergistic effect on tumors and cancer cells. Analogously, thepresent invention therefore also relates to use of the composition, asdisclosed herein, for the manufacture of a medicament for the treatmentof a cell growth disorder characterized by abnormal growth of human oranimal cells. For the purposes of the present invention, abnormal growthis characterized by uncontrolled cell division and/or differentiation.

In a more preferred embodiment, said cell growth disorder is cancer or agynaecological disorder characterized by abnormal growth of cells of thefemale mammal reproductive organs. The cancer referred to in the presentinvention is preferably one or more of basal cell carcinoma; biliarytract cancer; bladder cancer; bone cancer; brain and central nervoussystem cancer; breast cancer; cancer of the peritoneum; choriocarcinoma;connective tissue cancer; cancer of the digestive system (includingesophageal, stomach, colon, rectal or other gastrointestinal cancer);eye cancer; cancer of the head and neck; glioblastoma; hepaticcarcinoma; hepatoma; intra-epithelial neoplasm; kidney, adrenal, orrenal cancer; leukaemia; liver cancer; lung cancer (e.g., small-celllung cancer, non-small cell lung cancer, lung adenocarcinoma, and lungsquamous carcinoma); melanoma; myeloma; neuroblastoma; oral cavitycancer (e.g. lip, larynx, tongue, mouth or pharyngeal cancer);pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma;cancer of the respiratory system; salivary gland carcinoma; skin cancer;squamous cell cancer; testicular cancer; thyroid cancer; uterine,endometrial, cervical, vulval, ovarian or other gynaecological cancer;cancer of the urinary system; lymphoma including B-cell lymphoma,Hodgkin's and non-Hodgkin's lymphoma (NHL; including specific types suchas low grade/follicular, small lymphocytic, intermediategrade/follicular, intermediate grade diffuse, high grade immunoblastic,high grade lymphoblastic, high grade small non-cleaved cell, or bulkydisease NHL); mantle cell and AIDS-related lymphoma; chronic lymphocyticleukaemia; acute lymphoblastic leukaemia; Hairy cell leukaemia; chronicmyeloblastic leukaemia; as well as other carcinomas and sarcomas;post-transplant lymphoproliferative disorder (PTLD), as well as abnormalvascular proliferation associated with phakomatoses or oedema (such asthose that associated with brain tumors). More preferably, said canceris selected from one or more of bladder cancer; bone cancer; brain andcentral nervous system cancer; breast cancer; cancer of the digestivesystem (including esophageal, stomach, colon, rectal or othergastrointestinal cancer); cancer of the head and neck; glioblastoma;hepatic carcinoma; hepatoma; kidney, adrenal, or renal cancer;leukaemia; liver cancer; lung cancer; melanoma; myeloma; neuroblastoma;pancreatic cancer; prostate cancer; cancer of the respiratory system;skin cancer; testicular cancer; thyroid cancer; uterine, endometrial,cervical, vulval, ovarian or other gynaecological cancer; cancer of theurinary system; lymphoma or leukaemia. Still more preferably, saidcancer is selected from one or more of a carcinoma, glioma, melanoma orsarcoma, even more preferably melanoma, prostate cancer, colon cancer,breast cancer or pancreatic cancer, or alternatively melanoma orpancreatic cancer.

The female mammal reproductive organs referred to in the presentinvention are the female mammal sex organs located between the vaginaand falliopian tubes, inclusive. Said reproductive organs of the presentinvention are the uterus, fallopian tubes (comprising the utero-tubaljunction), ovaries, cervix and vagina, preferably the uterus, fallopiantubes, and cervix, more preferably the mammalian uterus. Therefore, thegynaecological disorder of the present invention is a disease whichaffects at least one cell of the female mammal reproductive tractlocated between the vagina and fallopian tubes, preferably the uterus,fallopian tubes and cervix. In the present invention, a gynaecologicaldisorder is more preferably a disease which affects at least one cell ina tissue of the mammalian uterus. In a most preferred embodiment, thegynaecological disorder is selected from endometriosis or leiomyoma.

In the present invention, the mammal is preferably hominine, bovine,equine, canine, feline, ovine, porcine, camelline, caprine or cervine.Furthermore preferably, said mammal is a human, dog, cow, horse orcamel, even more preferably a human or dog, and most preferably a human.

Thus, the BO-11X formulations can present a combination of componentsand other physico-chemical features that provide effects of medicalinterest (such as for treating of cancers such melanoma, or carcinomas)and that, preferably, can be obtained by applying a process compatiblewith regulatory and industrial requirements such as those applicable todrug manufacturing. In particular, BO-11X production stages are carriedout in strict compliance with the requirements of Good ManufacturingPractices in force in Europe (specifically Annex 13), USA, Japan, and/orother countries, and/or with the requirements for the approval ofInvestigational Medicinal Product Dossier (IMPD). BO-11X formulationsshould pass tests for sterility, purity, stability and biosafety(absence or substantially free of endotoxins, virus, bacteria,chemicals, metals, or other contaminants that are incompatible with usein humans). For instance, endotoxin level should be no more than 1.0EU/mg or 1 μg/mL, using officially approved detection kits.

Such components and features further characterizing BO-11X formulations,(as composition or particles comprised herein), may be one or more ofthe following ones:

(i) A composition comprising pharmaceutically acceptable carrier,excipient and/or adjuvant, with or without any further organic compound(such a solvent), inorganic compound, nucleic acid, aptamer, peptide orprotein having medical interest that is comprised in the particlesthemselves (for example, during their manufacturing) or later added intothe composition comprising these particles;

(ii) Particles formed by a polyalkyleneimine (such as linear PEI), inparticular having a weight average molecular weight between 17 and 23kDa, more preferably between 17.5 and 22.6 kDa;

(iii) A composition comprising particles formed by making a complexusing double-stranded polyribonucleotides at a concentration of at least0.5 mg/mL of the total (i.e. final) volume of said composition (forinstance, from 0.5 mg/ml up to 0.7 mg/mL, 0.9 mg/ml, 2 mg/mL or more);

(iv) Particles comprising a polyalkyleneimine and double-strandedpolyribonucleotides that are formed at a ratio of the number of moles ofnitrogen of said polyalkyleneimine to the number of moles of phosphorusof said double-stranded polyribonucleotide (i.e. the ratio of the numberof moles of nitrogen of said polyalkyleneimine to the number of moles ofphosphorus of said double-stranded polyribonucleotide used in formationof said particles) is equal to or greater than 2.5, more preferablybetween 2.5 and 5.5, still more preferably between 2.5 and 4.5,furthermore preferably between 2.5 and 3.5.;

(v) Particles having a mono-modal diameter distribution with anZ-average diameter, as defined herein and measured according to ISO22412, preferably below 200 nm, more preferably below 150 nm, and evenmore preferably in ranges comprised between 150 nm and 30 nm (such asbetween 150 nm and 50 nm, between 150 nm and 75 nm, between 100 nm and50 nm, between 150 nm and 100 nm, or between 60 nm and 130 nm);

(vi) Particles having a mono-modal diameter distribution, determinedfrom the diameter distribution according to ISO 22412, that isrepresented by D values D50% (the maximum particle diameter below which50% of sample intensity falls, also known as the median diameter)between 75 and 150 nm and D90% (the maximum particle diameter belowwhich 90% of sample intensity falls) between 140 and 250 nm;

(vii) At least 95% of particles have a diameter of 600 nm, preferably adiameter of 500 nm, more preferably a diameter of 400 nm, and even morepreferably a diameter of 300 nm. Yet more preferably, at least 90% ofparticles have a mono-modal diameter distribution below 300 nm;

(viii) A composition presenting an osmolarity comprised between 200 and600 mOsm/kg, preferably an aqueous composition comprising 5%(weight/volume) glucose presenting an osmolarity comprised between 200and 400 mOsm/kg, more preferably between 260 and 340 mOsm/kg;

(ix) A composition presenting a pH comprised between 2.7 and 3.4;

(x) An aqueous composition comprising 5% (weight/volume) D-glucosepresenting a specific optical rotation between +1500 and +3750degrees.mL·g⁻¹·dm⁻¹ at a wavelength of 589 nm at 20° C. at a wavelengthof 589 nm at 20° C. in water, referenced against water; and/or

(xi) A composition presenting a zeta potential equal or superior to 30mV, such as 38 mV, preferably comprised between 35 and 50 mV, or morepreferably comprised between about 40 and 45 mV (e.g. 43 mV), as definedherein and measured according to ISO 13099.

In a preferred embodiment, the present invention may relate tocompositions, in particular for pharmaceutical use, comprising particleswherein said particles comprise complexes of double-strandedpolynucleotides with a water-soluble, polycationic homo- orhetero-polymer wherein said particles are characterized by a mono-modaldiameter distribution that is defined by having a diameter of less thanor equal to 600 nm, preferably less than or equal to 300 nm and az-average diameter of less than or equal to 200 nm, preferably less thanor equal to 150 nm.

In another preferred embodiment, the double-stranded polyribonucleotidesare poly(I:C) molecules that are present in the BO-11X formulationsresult from the annealing of polyinosinic acid [poly(I)] molecules andpolycytidylic acid [poly(C)] single-stranded molecules that havethemselves specific ranges of percentages for sizes below 0.4 Kb,between 0.4 Kb and 0.85 Kb, between 0.85 Kb and 5.0 Kb, and above 5.0 Kbas indicated in the Examples which also provides means for generating anaqueous solution of poly(I:C) molecules (already containing or not anexcipient such as glucose or mannitol) and have appropriate features forbeing mixed with aqueous solution of a polyalkyleneimine (such aspolyethyleneimine) for producing the BO-11X formulations. Thepoly(I:C)-containing formulation resulting from mixing these two aqueoussolutions is then maintained as a batch preparation (preferably still asa aqueous solution or in a lyophilized form) or can be directly preparedin aliquots, each contained in a single-use vials, syringes, or otherappropriate container for storage, single use of such aliquots, and/orlyophilisation. BO-11X formulations (in a liquid or lyophilized form)can be stored at room temperature or a temperature below 0° C. or below−20° C.

In such preferred embodiment, further compounds (such as one or moreantibody, hormone, peptide, excipient, carrier, inhibitor of anenzymatic activity, chemotherapeutic agent, antibiotic, stabilizingagent, labelling agent, organic solvent, preservatives, carriers, orother drug) can be either added in each of the two aqueous solutions (ifnot altering the correct formation of the particles or any other of thefeatures listed above for BO-11X formulations) prior to their mixing orafter that BO-11X formulation has been produced by mixing the twoaqueous solutions (of double-stranded polyribonucleotide andpolyalkyleneimine). Such additional components that are consequentlyadministered at the same time with BO-11X components can provide acomposition with improvements in the bioavailability, efficacy,pharmacokinetic/pharmacodynamic profiles, stability, metabolization, orother property of pharmaceutical interest that are not observed wheneach of initial BO-11X formulation or the additional component (anothercompound of pharmaceutical interest, for instance) is administeredalone, or each of initial BO-11X formulation or the additional componentare administered separately.

In a further preferred main embodiment, the BO-11X formulation is foruse as a medicament, such as a pharmaceutical composition that isformulated (e.g. as an injectable, aqueous composition, optionallycomprising a pharmaceutically acceptable carrier, excipient and/oradjuvant) and administered for the delivery of double-strandedpolyribonucleotides to an organ or a tissue in a healthy state,presenting a disease related to a exogenous pathogenic agent (such abacteria or a virus), or presenting an alteration due to a cell growthdisorder characterized by abnormal growth of human or animal cells forinstance, due to cancer (that is, involving tumorogenic transformation,metastasis, toxic compound), or a gynaecological disorder characterizedby abnormal growth of cells of the female mammal reproductive organs).Thus, in a preferred embodiment, the present invention relates to amethod of treatment of a disease comprising administering thecomposition of the present invention to a human or animal. In a furtherpreferred embodiment, the present invention relates to a method oftreatment of a cell growth disorder characterized by abnormal growth ofhuman or animal cells, as defined herein, comprising administering thecomposition of the present invention to a human or animal.

Preferably, the BO-11X formulation is used in methods for inducing(directly or indirectly) the death of the tumor cell or suppress growthof the tumor cell, at scope of treating, reducing, ameliorating, orpreventing cancer growth, survival, metastasis, epithelial-mesenchymaltransition, immunologic escape or recurrence. More preferably, BO-11Xformulations are used in methods for treating solid tumors, such ascarcinomas, gliomas, melanomas, or sarcomas. In particular, the BO-11Xformulation is administered either systemically or more directly withinor in a location near to the tumor such as at the margin of the tumormass, in the surrounding epithelial cells, lymphatic or blood vessels(e.g. by intratumoral or peritumoral injection), or the abnormallygrowing cells of female mammal reproductive organs.

In some embodiments, the BO-11X formulation is the one producedaccording to the manufacturing methods, and then defined structurallyand functionally, as described in the Examples as BO-111 or BO-112formulations. The BO-11X formulation can further exhibit the biologicalactivities that were characterized for BO-110 as described inWO2011003883, namely activation of a family helicase MDA-5 or the levelof NOXA expression, in combination with the induction of autophagy incancer cells or in a cell line derived from cancer cells, preferablyfrom a human origin, albeit to an improved degree. Examples of celllines for validating BO-11X formulations are human SK-Mel-19, SK-Mel-28,SK-Mel-103 and SK-Mel-147 cells, and the murine B16 cells, said melanomacell lines presenting an increased expression of molecules such asInterferon Beta when exposed to a BO-11X formulation. Additionally, theBO-11X formulation presents no toxicity against normal cells that areused as controls, such as melanocytes or other skin cells, as well ascells of the immune system, which usually represent sites of secondarytoxicity in cancer treatment. The BO-11X formulation may also, followingthe autophagy and apoptosis of cancer cells (or any other effect oftherapeutic interest that this formulation may induce in such cells),induce the release of cancer cell antigens that may act as inducers of atumor-specific immunological response, in particular when BO-11Xformulation is administered locally to cancerous cells or tumors (e.g.by peritumoral or intratumoral injection, administering BO-11X at themargin of tumor mass, in surrounding epithelial cells, lymphatic orblood vessels, or directly within the tumor mass), with or without thesimultaneous or sequential administration of another drug or othertreatment for same indication.

The present invention also relates to a process to manufacture thecomposition, as disclosed herein, which comprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 500 nm to form sterilizedsolutions;

(iii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate of greater than or equal to 1 mL/min, or by simultaneous additionof each of said solutions into said mixing chamber, optionally byinjection, at a rate of greater than or equal to 1 mL/min; andoptionally

(iv) filtering the resulting aqueous composition through a filter havinga pore diameter of less than or equal to 600 nm to form a filtrate, orcentrifuging the resulting aqueous composition at greater than or equalto 22480 m/s² to form a supernatant; and/or

(v) lyophilising the resulting aqueous composition, filtrate orsupernatant.

Thus, in one preferred embodiment, the present invention may relate to aprocess to manufacture the composition, as disclosed herein, whichcomprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 500 nm to form sterilizedsolutions; and

(iiii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate of greater than or equal to 1 mL/min, or by simultaneous additionof each of said solutions into said mixing chamber, optionally byinjection, at a rate of greater than or equal to 1 mL/min; and

(iv) optionally lyophilising the resulting aqueous composition.

In addition, in another preferred embodiment, the present invention mayrelate to a process to manufacture the composition, as disclosed herein,which comprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 500 nm to form sterilizedsolutions;

(iiii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate of greater than or equal to 1 mL/min, or by simultaneous additionof each of said solutions into said mixing chamber, optionally byinjection, at a rate of greater than or equal to 1 mL/min; and

(iv) filtering the resulting aqueous composition through a filter havinga pore diameter of less than or equal to 600 nm to form a filtrate, orcentrifuging the resulting aqueous composition at greater than or equalto 22480 m/s² to form a supernatant; and

(v) optionally lyophilising the resulting filtrate or supernatant

More preferably, the process of the present invention does not comprisea final step of lyophilisation. In the process of the present invention,said double-stranded polyribonucleotide, said polyalkyleneimine and saidpharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant are as disclosed herein. Sterilizing each solution to formsterilized solutions takes place by independently filtering saidsolutions through a filter having a pore diameter of less than or equalto 500 nm, preferably by independently filtering said solutions througha filter having a pore diameter of less than or equal to 300 nm, morepreferably by filtering said solutions through a filter having a porediameter of less than or equal to 200 nm. Preferably the mixing of theresulting filtrates takes place through the large-scale convectivetransport of eddies and subsequently through elimination ofconcentration differences through purely diffusive transport. The mixingchamber may be any chamber or vessel in which the mixing of saidsolutions begins, such as a flask, reactor or mixer. More preferably,said mixing chamber has a fixed volume of between 0.1 and 20 mL,furthermore preferably between 0.2 and 10 mL, much more preferablybetween 0.5 and 8 mL. More preferably, mixing takes place by addition,optionally by injection, at a rate of between 1 mL/min and 2000 mL/min,still more preferably at between 10 and 1000 mL/min, furthermorepreferably at between 20 and 500 mL/min. Optional filtering of theresulting aqueous composition to form or collect a filtrate maysubsequently be performed through a filter having a pore diameter ofless than or equal to 600 nm, preferably not exceeding the diameter of500 nm, more preferably not exceeding the diameter of 400 nm, yet morepreferably not exceeding the diameter of 300 nm. Alternatively, optionalcentrifuging of the resulting aqueous composition to form or collect asupernatant may subsequently be performed at greater than 22480 m/s²(5000 rpm on a rotor having a radius of 0.082 m), preferably at greaterthan 27202 m/s² (5500 rpm on a rotor having a radius of 0.082 m), morepreferably at greater than 32372 m/s² (6000 rpm on a rotor having aradius of 0.082 m), yet more preferably 44062 m/s² (7000 rpm on a rotorhaving a radius of 0.082 m). In one especially preferred embodiment step(iv) is obligatory when the composition of the present invention is notachieved by steps (i) to (iii) of the process of the present invention,namely when addition is carried out at such that a rate that less than95% of the particles comprised in said aqueous composition has adiameter of less than or equal to 600 nm; and/or said particles have az-average diameter of greater than 200 nm, more preferably when saidparticles do not have a mono-modal diameter distribution. This may bethe case when addition is performed at a rate of between 1 mL/min and20mL/min, particularly in a reaction chamber of between 0.5 and 20 mL.Finally, the resulting aqueous composition, filtrate or supernatant maybe subjected to lyophilisation to afford the composition of the presentinvention as a particulate solid.

Thus, in one much more especially preferred embodiment of the process ofthe present invention, said process comprises

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 200 nm to form sterilizedsolutions;

(iiii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate between 20 mL/min and 100 mL/min, or by simultaneous addition ofeach of said solutions into said mixing chamber, optionally byinjection, at a rate between 20 mL/min and 100 mL/min, wherein saidmixing chamber has a volume of between 0.2 and 10 mL;

(iv) filtering the resulting aqueous composition through a filter havinga pore diameter of less than or equal to 500 nm to form a filtrate, orcentrifuging the resulting aqueous composition at greater than or equalto 32372 m/s² (6000 rpm on a rotor having a radius of 0.082 m) to form asupernatant; and

(v) optionally lyophilising the resulting filtrate or supernatant.

In another even much more especially preferred embodiment of the processof the present invention, said process comprises

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, wherein either or both solutions optionally further comprise apharmaceutically acceptable carrier, organic solvent, excipient and/oradjuvant;

(ii) independently filtering each solution through a filter having apore diameter of less than or equal to 200 nm to form sterilizedsolutions;

(iiii) mixing the resulting sterilized solutions in a mixing chamber toform an aqueous composition by addition of one of said solutions intothe other solution in said mixing chamber, optionally by injection, at arate between 30 mL/min and 100 mL/min, or by simultaneous addition ofeach of said solutions into said mixing chamber, optionally byinjection, at a rate between 30 mL/min and 100 mL/min, wherein saidmixing chamber has a volume of between 0.5 and 8 mL; and

(vi) optionally lyophilising the resulting aqueous composition.

In a further embodiment, the BO-11X formulation is produced according toa manufacturing process that involves the mixing of two aqueoussolutions, a first one comprising the double-strandedpolyribonucleotides (or a salt or solvate thereof) and a second onecomprising the polyalkyleneimine (or a salt or solvate thereof) so thatthe resulting particles present the features defined above, inparticular with respect to the diameter and the mono-modal diameterdistribution as well the appearance as an essentially clear colloidalsolution.

As described with further details in the Examples, the BO-11Xformulations can be provided by filtering and/or centrifugingpharmaceutical composition comprising particles formed bydouble-stranded polyribonucleotides and a water-soluble, polycationichomo- or hetero-polymer, providing the BO-11X formulations as bulk orsingle use liquid compositions without visible aggregates. Such aprocess to manufacture the composition comprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, optionally together with a pharmaceutically acceptable carrier,organic solvent, excipient and/or adjuvant present in either solution;

(ii) mixing said solutions; and

(iii) filtering the resulting mixture through a filter having a porediameter of less than or equal to 600 nm, or centrifuging at greaterthan 22480 m/s².

This process can be further adapted for the actual components of thecomposition (double-stranded polyribonucleotides, the polyalkyleneimine,and optional further components) as well as the desired methods ofusing, storing, shipping, packaging, and/or administering thecomposition, in particular if the composition requires to bemanufactured immediately prior to the use or, as is more common forpharmaceutical compositions, manufactured for long-term storage and/orin the form of multiple containers each one for a single-use (e.g. insterile vials or syringes), and containing particles having the mostuniform size, poly(I:C) content, stability, solubility, and, finally,biological effects when administered.

At this scope, the mixing and filtering steps (ii) and (iii) above canbe adapted at the level of order of filtering and/or mixing, method ofmixing, the mixing speed, and/or the amount of solutions that is mixed.In a further preferred embodiment, the process to manufacture BO-11Xformulations comprises:

(i) preparing an aqueous solution of at least one double-strandedpolyribonucleotide, or a salt or solvate thereof, and an aqueoussolution of at least one polyalkyleneimine, or a salt or solvatethereof, optionally together with a pharmaceutically acceptable carrier,organic solvent, excipient and/or adjuvant present in either solution;

(ii) sterilizing each solution by filtering them independently;

(iii) mixing said solutions in the container for storing, lyophilising,and/or using the composition by adding either solution first and thenadding the other solution at an injection speed superior to 50 rpm at aflow speed between 1 mL/min and 50 mL/min; and

(iv) sealing the container.

Combinations

A composition according to the present invention can be used incombinations including other compounds or treatments with which it isknown to be compatible, if not providing an additive or even synergisticeffect. For instance, poly(I:C) molecules can be used in combinationwith different anti-cancer drugs, antibodies, radiotherapy, orchemotherapy (Le U et al., 2008; Le U et al., 2009; Taura M et al.,2010; Matijević T et al., 2011; Levitzki A, 2012; Yoshino H andKashiwakura I, 2013; Hefner A et al., 2013) or different length ofpoly(I:C) molecules (Zhou Y et al., 2013). Poly(I:C) molecules have alsobeen used as an adjuvant or synergically-acting agent when combined withother agents such as in vaccination with cancer antigens or cell lysates(Ammi R et al., 2015), agents blocking PD-1/PD-L1 pathway (Nagato T andCelis E, 2014), other TLR agonists, such as TLR9 agonist CpG ODN (ZhangY et al., 2014), dichloroacetate (Ohashi T et al., 2013), IL27 (ChibaYet al., 2013), kinase inhibitors such as sorafenib (Ho Vet al., 2015),proapoptotic proteins such as NS1 (Gupta S et al. 2016), Zoledronic acid(Chen L et al., 2013), or all-trans retinoic acid (Szabo A et al.,2012). Other uses of BO-11X formulations may become apparent in view ofactivities of poly(I:C) molecules towards specific cell types recentlydemonstrated, at least using in vitro assays, such as on pre-adipocytes,inhibiting differentiation and differentiation in adipocytes (Yu L etal., 2016), mesenchymal stem cells, enhancing immunosuppressive effects(Cho K. et al., 2016; Vega-Letter A et al., 2016), or activation of NKcells (Perrot I et al., 2010).

In some aspects, the present invention relates to a pharmaceuticalcomposition comprising an effective amount of a BO-11X formulation andan effective amount of one or more immune-modulating agents, inparticular agents that target immune checkpoint molecules (such as PD-1,PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3,B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1, SHP-2, PPP2R5A orOX-40), said compounds commonly named as checkpoint inhibitors (CPIs).

Accordingly, the present invention provides compositions and methodsthat are useful in combination therapies and regimens comprising theadministration of BO-11X formulations and another therapeutic agent ortreatment (including radiotherapy, chemotherapy, cryotherapy, tumorablation, or photodynamic therapy). In particular, the present inventionrelates to a method for treating, ameliorating, or preventing cancergrowth, metastasis, ulceration, immunologic escape or recurrence in asubject, comprising administering a BO-11X formulation and one or moreanticancer drug, preferably an immune-modulating agent, wherein theadministration is simultaneous (as separate formulations or in thecontext of a co-formulation) or sequential (in any order or inconsecutive cycles of administration). In some aspects, the presentinvention relates to a method for treating cancer, comprisingadministering an effective amount of BO-11X formulation agent and aneffective amount of one or more immune-modulating agents to a subject inneed thereof, in particular wherein the subject is undergoing cancertherapy with one or more immune-modulating agents.

In various embodiments, the immune-modulating agent is preferably anantibody including a monoclonal antibody and other antibody formats, orany other pharmaceutically available agent that binds a cell surfaceprotein that control immune response, thus acting as a CPI, This CPI canblock, reduce and/or inhibit PD-1 and PD-L1 or PD-L2 and/or the bindingof PD-1 with PD-L1 or PD-L2. Alternatively, this CPI can block, reduceand/or inhibit reduces and/or inhibits the activity of other immunecheckpoint molecules such as CTLA-4, AP2M1, CD80, CD86, SHP-2, and/orPPP2R5A As a further alternative, the CPI increases and/or stimulatesCD137 (4-1BB) and/or the binding of CD137 (4-1BB) with one or more of4-1BB ligand and TRAF2.

In various embodiments, the methods involving combination therapies andregimens for the treatment of cancer comprising the administration ofBO-11X formulations may further defined with respect to a specificadministration method, wherein the BO-11X formulation is administered bya route different form the one of the other therapeutic compound, suchas an immune-modulating agents, and preferably a CPI. This method mayinvolve administering the BO-11X formulation by intratumoral orperitumoral injection (within the tumor, at the margin of the tumormass, in the surrounding epithelial cells, lymphatic or blood vessels)or other means that allow administering the BO-11X formulation directlywithin or in proximity of cancer cells or organ comprising the cancercells (and not indirectly, for instance through bloodstream) and thesystemic administration of CPI or other immunostimulatory agent). TheBO-11X formulation by intratumoral or peritumoral injection may beperformed at the level of skin, i.e. into the skin (e.g. for treatingmelanoma or in connection to the combination with a vaccine) or of aninternal organ or tissue, i.e. into said internal organ or tissue (e.g.by intrahepatic injection for treating liver cancer or intravesicularadministration for bladder cancer). Such local administration of BO-11Xformulation preferably follows or (preferably) is followed by theadministration of the immunostimulatory agent.

Additional Effects and Uses

In a further embodiment, the present invention provides pharmaceuticaluses and methods involving the administration of a BO-11X formulationfor increasing immune response against a pathogen or undesirablebiological agent and in particular for enhancing an anti-tumor immuneresponse, potentially acting itself as an immune-modulating agent. Suchan effect can be monitored by measuring tumor-related immune responseinto the tumor site and tumor microenvironment (or in the bloodstream,other biological fluids, and tissues) at the level of relevant celltypes or subpopulations (e.g. dendritic cells, T regulatory cells, Tcells and/or NK cells) and/or of immunological biomarkers (e.g.chemokines, growth factors, cytokines, and their receptors).

Pharmaceutical Compositions

Also provided are methods for making a pharmaceutical composition ofBO-111 or BO-112 (and/or other BO-11X formulations) by mixing a BO-11Xformulation and one or more pharmaceutically acceptable adjuvant,diluent, carrier, or excipient thereof. Such components can be adaptedfor the specific medical indication (e.g. a solid cancer or ahematological cancer) and/or the administration means e.g. by injection(peritumoral, intratumoral, intrahepatic, intrapancreatic, orsubcutaneous injection), by inhalation, or orally.

BO-11X formulation-based combination treatments may involve the same ordifferent administration routes for BO-11X formulation and the othercompound. In particular, the BO-11X formulation can be administered, asfor other immunostimulatory RNA agents, by intratumoral or peritumoralinjections that may activate the immune system prior to the systemicadministration of a therapeutic antibody acting as CPI such a PD-1/PD-L1pathway inhibitors (Bald T et al., 2014) or activated T cells (Amos S Met al., 2011). Alternatively, the BO-11X formulation together with othertherapeutic compounds being TLR agonist or ligands such as CpG moleculesor Resiquimod for enhancing the effect of anticancer vaccination(Sajadian A et al., 2014) or Dendritic cells (Fujimura T et al., 2006).

Immune-Modulating Agents

The BO-11X formulation may be combined with one or moreimmune-modulating agents. In some embodiments, the immune-modulatingagent is a co-stimulatory or co-inhibitory molecule (e.g. of one or moreimmune cells, such as, by way of non-limitation, T cells and NK cells).In some embodiments, the immune-modulating agent is an agent thatmodulates a CD4 and/or CD8 T cell, for instance by acting as agonist orantagonist with respect to CD3, CD4, CD8, PD-1, PD-L1, PD-L2, CTLA-4,CD137, CD96, CD73, CD90, CD47, CD69, CD26, TIM3, and LAG3. In otherembodiments, the immune-modulating agent is an agent that modulates NKcells, for instance by acting as agonist or antagonist with respect toCD3, NKp46, CD16, NKG2D, NKp44, and NKp30. In other embodiments, theimmune-modulating agent is an agent that modulates tumor stroma andendothelium biomarkers, for instance by acting as agonist or antagonistwith respect to CD45, PD-L1, PD-L2, PTEN, and CD31.

The immune-modulating agent is provided as a further compound in form ofa chemical organic or inorganic compound, a nucleic acid, an aptamer, apeptide, a protein, and more particularly an antibody that binds therelevant target in the biological fluids or on cell surface. Theantibody may be polyclonal or monoclonal; intact or truncated (e.g.,F(ab′)₂, Fab, Fv); bispecific or multispecific; xenogeneic, allogeneic,syngeneic, or modified forms thereof (e.g., a chimeric antibody or ahumanized antibody). When the immune-modulating agent is a monoclonalantibody, it may be a non-human mammal-derived monoclonal antibody, arecombinant chimeric monoclonal antibody, a recombinant humanizedmonoclonal antibody, or a human monoclonal antibody.

The antibody that acts as immune-modulating agent can further comprisethe structural elements normally required for exerting the requiredbiological activity, such as four polypeptide chains, two heavy (H)chains and two light (L) chains inter-connected by disulfide bonds thatare capable of binding one or more antigens (e.g. bi-specific ormulti-specific antibodies) and presenting an Fc region of animmunoglobulin (e.g. IgA, IgG, IgE, IgD or IgM) which may interact withFc receptors and activate an immune response leading to depletion and/orcell death of immune cells or other cells. Each heavy chain is comprisedof a heavy chain variable region (V_(H)) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH₁, CH₂ and CH₃. Each light chain is comprised of a light chainvariable region (V_(L)) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each variable region (V_(H) or V_(L)) contains 3 CDRs,designated CDR1, CDR2 and CDR3. Each variable region also contains 4framework sub-regions, designated FR1, FR2, FR3 and FR4. The termantibody includes all types of antibodies, including, for example, IgA,IgG, IgD, IgE and IgM, and their respective subclasses (isotypes), e.g.,IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. An antibody, in someembodiments, also refers to antibody fragments and antigen-bindingfragments.

Antibodies suitable for practicing the methods described herein can beof various antibody formats, for example, monoclonal, polyclonal,bispecific, multispecific, and can include, but are not limited to,human, humanized or chimeric antibodies, comprising single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, and/or binding fragments of any of the above.Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain at least two antigens or target binding sites against at leasttwo targets described herein. The immunoglobulin molecules describedherein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class(e.g. IgGI, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. In addition, antibodies (e.g. mono-, bi-,and/or multi-specific) suitable for practicing the invention describedherein can be provided in any of the alternative formats that aredisclosed in the literature, for example Diabodies; Flexibodies; CamelidAntibodies, Immunobodies; Triomabs, Pepbodies, Vaccibodies, minibodies,Fcabs, UniBodies, or DuoBodies (Storz U 2011).

PD-1 (also known as CD279 or Programmed cell death protein 1) is amember of the B7 family of receptors. In some embodiments, PD-1 refersto the human PD-1 sequence (see, e.g. NCBI Reference Sequence:NP_005009) and any naturally occurring allelic, splice variants, andprocessed forms thereof (Keir M et al., 2008; UniProt: Q15116). PD-1binds PD-L1 (also known as CD274 or B7-H1) and PD-L2 (also known asCD273 or B7-DC), which are also members of the B7 family. In someembodiments, PD-L1 refers to human PD-L1 (see, e.g. GenBank: AF233516),PD-L2 refers to human PD-L2 (e.g. NCBI Reference Sequence: NM_025239),together with and any naturally occurring allelic, splice variants, andprocessed forms thereof.

In various embodiments, the immune-modulating agent targets one or moreof PD-1, PD-L1, and PD-L2. In various embodiments, the immune-modulatingagent is PD-1 inhibitor. In various embodiments, the immune-modulatingagent is an antibody specific for one or more of PD-1, PD-L1, and PD-L2.For instance, in some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, nivolumab,(ONO-4538/BMS-936558, MDX1106, OPDIVO), pembrolizumab (KEYTRUDA),pidilizumab (CT-011), MK-3475, BMS 936559, MPDL3280A.

In some embodiments, the BO-11X formulation is combined with one or moreof BMS-936559 and MEDI4736 for treatment of, for example, advanced solidtumors. In some embodiments, the BO-11X formulation is combined with oneor more MPDL3280A (optionally with vemurafenib) and MEDI4736 (optionallywith one or more of dabrafenib and trametinib) for the treatment ofmelanoma. In some embodiments, the BO-11X formulation is combined withone or more MPDL3280A (optionally with erlotinib) and MEDI4736(optionally with tremelimumab) for the treatment of NSCLC. In someembodiments, the BO-11X formulation is combined with MPDL3280A(optionally with one or more of bevacizumab and sunitinib) for thetreatment of RCC. In some embodiments, the BO-11X formulation iscombined with MPDL3280A for the treatment of solid or hematologicalmalignancies. In some embodiments, the BO-11X formulation is combinedwith one or more MPDL3280A (optionally with one or more of bevacizumab,chemotherapy and cobimetinib); MEDI4736 (optionally with tremelimumab)and MSB0010718C for the treatment of solid tumors. In some embodiments,the BO-11X formulation is combined with AMP-224 for the treatment ofadvanced cancer. In some embodiments, the BO-11X formulation is combinedwith nivolumab (optionally with iliolumbar (anti-KIR)) for the treatmentof advanced solid tumors. In some embodiments, the BO-11X formulation iscombined with nivolumab for the treatment of castration-resistantprostate cancer, melanoma, NSCLC, and RCC. In some embodiments, theBO-11X formulation is combined with pembrolizumab for the treatment ofcolon cancer. In some embodiments, the BO-11X formulation is combinedwith pembrolizumab for the treatment of gastric cancer, head and neckcancer, TNBC, and urothelial cancer. In some embodiments, the BO-11Xformulation is combined with nivolumab (optionally with ipilimumab) forthe treatment of gastric cancer, pancreatic cancer, small-cell lungcancer, and TNBC. In some embodiments, the BO-11X formulation iscombined with nivolumab (optionally with ipilimumab) for the treatmentof glioblastoma. In some embodiments, the BO-11X formulation is combinedwith nivolumab for the treatment of hepatocellular cancer. In someembodiments, the BO-11X formulation is combined with pembrolizumab forthe treatment of Hodgkin lymphoma, myeloma, myelodysplastic syndrome,and non-Hodgkin lymphoma. In some embodiments, the BO-11X formulation iscombined with pidilizumab for the treatment of malignant gliomas. Insome embodiments, the BO-11X formulation is combined with one or more ofnivolumab (optionally with one or more of ipilimumab, and multiple class1 peptides and montanide ISA 51 VG; and optionally sequentially withipilimumab) and pembrolizumab for the treatment of melanoma. In someembodiments, the BO-11X formulation is combined with pembrolizumab forthe treatment of melanoma and NSCLC. In some embodiments, the BO-11Xformulation is combined with one or more of nivolumab (optionally withone or more of gemcitabine/cisplatin, pemetrexed/cisplatin,carboplatin/paclitaxel, bevacizumab, erlotinib, and ipilimumab) andpembrolizumab for the treatment of NSCLC. In some embodiments, theBO-11X formulation is combined with pidilizumab (optionally withgemcitabine) for the treatment of pancreatic cancer. In someembodiments, the BO-11X formulation is combined with pidilizumab(optionally with one or more of sipuleucel-T and cyclophosphamide) forthe treatment of prostate cancer. In some embodiments, the BO-11Xformulation is combined with one or more of nivolumab (optionally withone or more of sunitinib, pazopanib, and ipilimumab), pembrolizumab(optionally with pazopanib), and pidilizumab (optionally with dendriticcell/RCC fusion cell vaccine) for the treatment of RCC. In someembodiments, the BO-11X formulation is combined with one or more ofanti-LAG3 (BMS-986016, optionally with nivolumab), nivolumab (optionallywith interleukin-21), and AMP-554 for the treatment of solid tumors. Insome embodiments, the BO-11X formulation is combined with pembrolizumabfor the treatment of solid tumors.

In various embodiments, the immune-modulating agent targets one or moreof CD137 or CD137L. In various embodiments, the immune-modulating agentis an antibody specific for one or more of CD137 or CD137L. Forinstance, in some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, urelumab (also known asBMS-663513 and anti-4-1BB antibody). In some embodiments, the BO-11Xformulation is combined with urelumab (optionally with one or more ofnivolumab, lirilumab, and urelumab) for the treatment of solid tumorsand/or B-cell non-Hodgkins lymphoma and/or head and neck cancer and/ormultiple myeloma. In some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, ipilimumab (MDX-010,MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). In some embodiments,the BO-11X formulation is combined with ipilimumab(optionally withbavituximab) for the treatment of one or more of melanoma, prostatecancer, and lung cancer.

In various embodiments, the immune-modulating agent targets CD20. Invarious embodiments, the immune-modulating agent is an antibody specificCD20. For instance, in some embodiments, the immune-modulating agent isan antibody such as, by way of non-limitation, Ofatumumab, obinutuzumab(GAZYVA), AME-133v, Ocrelizumab, TRU-015, and veltuzumab.

Validation of BO-11X Formulations

The pre-clinical validation of therapeutic efficacy of a BO-11Xformulation (in accordance to the present invention and as exemplifiedby BO-111 and BO-112 in the Examples) can be performed in cell-basedassays and, most interestingly, in animal models where differentexperimental criteria can be tested and compared to establish the mostappropriate conditions to achieve therapeutic effects effectively, usingthe BO-11X formulation alone or in combination with a candidate orapproved anti-cancer drug, These criteria include the doses,administration route, the order and/or the frequency of administrationof either compound at the scope to identify which are the betterconditions for therapeutic use of a BO-11X formulation (alone orsynergically with a candidate or approved anti-cancer drug) in terms ofefficacy, safety, and/or clinical use.

The effects of different dosage of a BO-11X formulation, number and/orsite of administration (in particular, by injecting it in one or moresites), route of administration, frequency, and/or time point foradministration can be associated to relevant end-points andphysiological parameters that are measured in biological samplesobtained from cells or (preferably) animals that are exposed to thetested compounds, alone or in combination with other drugs. Anon-limiting list of such parameters includes regression of tumor size,block of tumor growth and/or proliferation of tumor cells, apoptosis,reduced tumor vascularization or metastasis, overcoming resistance to acommon anti-cancer drug (or otherwise improving the response to such adrug in the treated population), reduced treatment related-adverseevents on normal tissues and functions, modulation of immune responseand/or of immune cells having specific activities and features,identification of biomarkers or specific cell populations in biologicalmaterials (e.g. present in cancer cell preparations, tumor biopsies orbiological fluids) whose increase (or decrease) is known in theliterature as being associated to anti-cancer effect in general, and inparticular to survival of animal models and possibly of cancer patients.Whenever possible, such end-points are measured at intermediate andfinal time-points following the administration of each test compound, ora test combination of compounds at a given dose and/or regimen, by usinga specific route of administration and/or pharmaceutical formulations.

The therapeutic, anti-cancer efficacy of a BO-11X formulation can betested alone or in combination with standard-of-care, conventionaltreatments (such as radiotherapy, chemotherapy, inhibitors of cellularkinases, etc.) or treatments involving novel mechanisms and/or novelcandidate anti-cancer drugs. Indeed, a category of novel anti-cancercompounds that can be tested in combination with a BO-11X formulationare those improving the anti-tumor immune responses within tumormicroenvironment, by providing a systemic antitumor immunity thattargets disseminated tumor cells are eliminated. This approach has beenproven successful in animal models and patients and can improve theoutcome of conventional therapies such as radiotherapy or chemotherapy(see, for example, Galluzzi L et al., 2014; Vacchelli E et al., 2013;Van der Jeught K et al., 2015).

This growing panel of new cancer drugs targets the mechanisms by whichcancer cells escape from immune detection and destruction by human body.Cancer-specific immunotherapies may provide a series of advantages whencompared to other cancer therapies (e.g. tumor cell specificity).Indeed, the identification of a series of molecular targets for cancerimmunotherapies is allowing major advances in defining the mechanismsand compounds that can provide the appropriate co-stimulatory (orco-inhibitory) effect on immune responses against tumors with respect totheir plasticity, heterogeneity, resistance, or microenvironment.

A non-limiting list of cancer passive or active immunotherapies, eachacting on different molecular targets and/or mechanisms, includestumor-targeting or other immunomodulatory monoclonal antibodies,oncolytic viruses, immunostimulatory cytokines, adoptive cell transferand other cell-based therapies, DNA- or peptide-based vaccines,inhibitors of immunosuppressive metabolism, or agonists of patternrecognition receptors. Among these mechanisms, molecular targets againstwhich antibodies or other compounds having either agonistic orantagonistic activity (depending on their role in immune response orcancer escape from immune response) include a series of cell membraneproteins such as PD-1, PD-L1, PD-L2, CTLA-4, CD137, CD38, OX-40, CD26,TIM3, and LAG3 that are checkpoints for tumor development and againstwhich different agonistic or antagonistic antibodies are availablecommercially or in scientific repositories for characterizing theirspecificity and/or level of anti-cancer effect against the human antigenor (when dealing with animal models) the corresponding rodent antigen.

Despite impressive patient responses to agents targeting theseco-stimulatory or co-inhibitory molecules (e.g. therapies that are basedon an anti-PD-1 or anti-PD-L1 antibody) the clinical response to immunecheckpoint inhibitors is still incompletely or inefficiently achievingthe desired therapeutic effect in too many cancer patients. A BO-11Xformulation in an appropriate combination with a compound such as ananti-PD-1 or anti-CD137 antibody may enhance the reduction of tumorgrowth and metastasis (or increase the number of subjects presentingsuch reduction), when compared to the effect of the administration ofone of those two anti-cancer agents alone, possibly beyond the additiveeffects that may be expected.

The therapeutic effects of a BO-11X formulation can be evaluated in oneof the several cell-based models that are based on isolated or mixedcell culture including primary cancer cells or established cancer celllines, preferably from a human origin. Examples of cancer cell lines forvalidating BO-11X formulations can be defined according to cancer typesuch as melanoma (human SK-Mel-19, SK-Mel-103 and UACC62 cells; murineB16 cells), carcinoma (mouse Hepa 1-6 cells; rat FAO cells), breastcancer (human BT483, HCC1143, HCC1937, MDA-MB-231, MDA-MB-415,MDA-MB-468, and BT549 cells), pancreatic cancer (human MiaPaCa2,IMIM-PC2, Panc1, Panc0203, Panc 3.27, or BxPc3 cells), or other relevantcell lines that are available through ATCC, other official or academicrepositories, or commercial providers. The anticancer effects of BO-11Xformulations can be evaluated at the level of period of time, frequency,and/or dose that is required to have a block of proliferation, thedeath, the expression of biomarkers, and/or the release of signalingmolecules (such as chemokines or Interferon Beta) that indicate apotentially relevant effect of the BO-11X formulation to be confirmed inmore physiological conditions.

Then, the effects of a BO-11X formulation can be evaluated in tumoranimal models in which the anti-tumor response due to the administrationof an exemplary formulation such as BO-112 is assessed in differentprotocols for both monotherapy and combination treatment (e.g. togetherwith a CPI such as an anti-PD-1 antibody) throughout a shorter or longerperiod of time after administration. The study may be pursued byadministering BO-112 and/or anti-PD-1 antibody in animals at a giventime of tumor development due to proliferation of injected cells, thatis after a specific number of days following the injection of cancercells or (preferably) that present the desired tumor size (e.g. anaverage size of 80-100 mm³), or even following its disappearance (forevaluate any effect of each drug or drug combination on tumor relapse).The study would also involve control compounds that are either negative(e.g. vehicle alone) or positive controls, such as chemotherapeutic orother anti-cancer drugs that are indicated in the literature as standardfor drug effectiveness for a specific tumor and/or in a given animalmodel. These activities of validation in animal models and animal cellmay also lead to the development of BO-11X formulation for veterinaryuse.

The animal model is typically a mouse model in which the cancer isconsequent to either the transfer and engraftment of human cancer cells(coming from an immortalized cell line or a cancer biopsy that isobtained by a patient) or the induction (or transfer) of mouse tumorcells in the animals. Cells can be originated by different type oftumors (e.g. lung carcinoma, melanoma, lymphoma) and can be injectedsub-cutaneously in the flank of the mice to simply the detection oftumor and the analysis of its size and/or composition during the study.Mice are then treated by randomizing them into groups each of a sizeallowing statistical analysis of results (e.g. 3, 5, 10, 12, 15, 20 ormore animals for each control or treatment group).

Features that a BO-11X formulation such as BO-112 and a checkpointinhibitor such as an anti-PD-1 antibody may improve in cancer animalmodels (in particular when appropriately combined in terms of amount,order, or other administration criteria), may include animal survivalafter treatment and/or tumor disappearance, reduced tumor relapse,limited or delayed toxicity and/or resistance effects, and response tore-challenge of tumor inoculation after termination of the treatmentwith BO-112 and/or anti-PD-1 antibody.

The exemplary BO-11X formulation that is identified structurally andfunctionally above as BO-112 and anti-PD-1 antibody can be administered(alone or in combination, in single or multiple doses) at differentlocations with respect to tumor cells and/or in different amount.Typically, BO-112 and the monoclonal antibody specific for mouse PD-1(e.g. clone RMP1-14 from BioLegend or similar ones available by otherproviders) are injected sub-cutaneously, intravesicularly,intraperitoneally, peritumorally or intratumorally (depending on themodel and tumor molecular and pathological features) at a concentrationthat is determined with respect to animal weight (e.g. between 0.01 and2.5 mg/kg), concentration in the injected volume (e.g. between 0.01 and0.5 mL, and/or content in each dose (e.g. between 0.01 and 250 μg perdose). In particular, the dose-response of BO-112 in differentconcentrations, combined with a fixed anti-PD-1 antibody dose (or viceversa), may allow determining any advantageous effect that is consequentto the significant reduction in the amount of either compound that isadministered due to the combination with the other compound (e.g.unaffected or even improved efficacy and/or safety profile; abscopaleffects in tumors that are in different, untreated locations).

BO-112 can be injected in one or multiple cycles (e.g. 2, 3, 4, or more)that are separated by given number of days (1, 2, 3, 5, 7, or more).Alternatively, when BO-112 is co-administered with the anti-PD1antibody, BO-112 can be injected immediately before (or after) theantibody (or in a single injectable preparation), again in one ormultiple cycles (that are separated by given number of days. Stillalternatively, the two agents may be formulated, or administered in anysequential order, but separated by variable period of time (e.g. 1 hour,3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days ormore). In particular, when BO-112 is administered after anti-PD-1antibody, its later administration (alone or further in combination withthe anti-PD-1 antibody) may provide an anti-tumor rescue effect inanimals in which anti-PD-1 antibody was ineffective against tumor cells,overcoming any specific tumor resistance or escape mechanism. At the endof treatment, all surviving animals can be left untreated for 1, 2, 3,or more consecutive weeks to monitor if and how tumor cells reappear,with or without re-challenging all animals that had a completeregression of tumor with a further subcutaneous injection of cancercells.

The effects of BO-112, alone or in combination with the anti-PD1antibody as listed above, can be assessed as interim results that arereported during the study without sacrificing the animals (e.g. bymeasuring tumor size, percentage of mice still alive, bodyweight, orbehavioural criteria) or after sacrificing the animal (or in alreadydead mice) for determining molecular features of tumor and/or normalcells (including total number and/or specific sub-populations of NKcells, tumor-infiltrated lymphocytes, splenocytes, incorporation ofradiolabeled precursors, and other cells that may be involved in theanti-tumor local or systemic immune responses, such as. Myeloid-derivedsuppressor cells (MDSCs), regulatory T cells (Tregs); tumor associatedneutrophils (TANs), M2 macrophages, and tumor associated macrophages(TAMs). I parallel, the presence/absence of relevant biomarkers can bedetermined by PCR amplification of relevant RNAs, or at protein level onthe surface of cells within tissues or circulating in blood, such ascytokines or chemokines by using standard immunological assays and kits.

This global phenotype analysis can be performed by using cells isolatedfrom tumors, blood, spleen, lymph nodes, or other relevant tissues andlocations, for detecting any statistically and/or therapeuticallyrelevant change in the number of cells expressing cell surface markersthat are detected by flow cytometry and described in the literature suchas CD3, CD4, CD25, FoxP3, CD8, PD-1, PD-L1, PD-L2, PTEN, CTLA-4, CD137,CD96, CD73, CD90, CD47, CD69, CD26, TIM3, LAG3, Gr1, CD11b, Ly6C, Ly6G,NKp46, CD16, NKG2D, NKp44, NKp30, CD45, and CD31. Such cells can be alsoevaluated at the level of tumor antigen- specific immune response,expression of relevant transcription factors, cytokines or chemokines(e.g. IFN-gamma, IFN-beta, TNFalpha, HIF1a, HIF2a, p53), or by usingother cell-based assays.

Additionally, macroscopic examination of organs and skin andmicroscopic, pathological analysis in either immune-deficient fullyimmune competent animal models can further provide indication about theefficacy of the study compounds (alone or in the combination) or oftheir toxicity, such as organ inflammation and necropsy. Thequantitative data that are generated in similar studies can be comparedamong the different experimental groups by using the appropriatestatistical tests, with and without corrections for multiple testing, atthe scope to evaluate which therapeutic (in particular anti-tumor)effects are provided by the administration of a BO-11X formulation,alone or in combination with another anti-cancer agent.

Methods of Treatment and Patient Selections

In some embodiments, the present invention relates to a method fortreating, reducing, ameliorating, or preventing cancer growth, survival,metastasis, epithelial-mesenchymal transition, immunologic escape orrecurrence, comprising administering by administering a BO-11Xformulation and one or more immune-modulating agents. The cancer may bean oncological disease. The cancer may be a dormant tumor, which mayresult from the metastasis of a cancer. The dormant tumor may also beleft over from surgical removal of a tumor. The cancer recurrence may,for example, be tumor regrowth, a lung metastasis, or a livermetastasis.

In various embodiments, the cancer is one or more of basal cellcarcinoma, biliary tract cancer; bladder cancer; bone cancer; brain andcentral nervous system cancer; breast cancer; cancer of the peritoneum;choriocarcinoma; connective tissue cancer; cancer of the digestivesystem (including esophageal, stomach, colon, rectal or othergastrointestinal cancer); eye cancer; cancer of the head and neck;glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney, adrenal, or renal cancer; leukemia; liver cancer; lung cancer(e.g. small-cell lung cancer, non-small cell lung cancer, lungadenocarcinoma, and lung squamous carcinoma); melanoma; myeloma;neuroblastoma; oral cavity cancer (lip, larynx, tongue, mouth, andpharynx); pancreatic cancer; prostate cancer; retinoblastoma;rhabdomyosarcoma; cancer of the respiratory system; salivary glandcarcinoma; skin cancer; squamous cell cancer; testicular cancer; thyroidcancer; uterine, endometrial, cervical, vulval, ovarian or othergynecological cancer; cancer of the urinary system; lymphoma includingB-cell lymphoma, Hodgkin's and non-Hodgkin's lymphoma (NHL; includingspecific types such as low grade/follicular, small lymphocytic,intermediate grade/follicular, intermediate grade diffuse, high gradeimmunoblastic, high grade lymphoblastic, high grade small non-cleavedcell, or bulky disease NHL), mantle cell and AIDS-related lymphoma;chronic lymphocytic leukemia; acute lymphoblastic leukemia; Hairy cellleukemia; chronic myeloblastic leukemia; as well as other carcinomas andsarcomas; post-transplant lymphoproliferative disorder (PTLD), as wellas abnormal vascular proliferation associated with phakomatoses or edema(such as those that associated with brain tumors).

In various embodiments, the cancer is a biliary tract cancer. In someembodiments, the biliary tract cancer is selected from pancreaticcancer, gallbladder cancer, bile duct cancer, and cancer of the ampullaof Vater. In various embodiments, the cancer is liver cancer. In variousembodiments, the cancer is colon cancer. In some embodiments, thebiliary tract cancer is cholangiocarcinoma and/or an adenocarcinoma.

In some embodiments the BO-11X formulation and/or immune-modulatingagent is used to treat cancers of various stages (e.g. Stage I, or II,or III, or IV). By way of non-limiting example, using the overall stagegrouping, Stage I cancers are localized to one part of the body; StageII cancers are locally advanced, as are Stage III cancers. Whether acancer is designated as Stage II or Stage III can depend on the specifictype of cancer. In one non-limiting example, Hodgkin's disease, Stage IIindicates affected lymph nodes on only one side of the diaphragm,whereas Stage III indicates affected lymph nodes above and below thediaphragm. The specific criteria for Stages II and III therefore differaccording to diagnosis. Stage IV cancers have often metastasized, orspread to other organs or throughout the body.

In some embodiments, the BO-11X formulation (and/or theimmune-modulating agent) reduces side effects of the therapies that apatient may experiences. For example, the combination therapy of anBO-11X formulation and one or more immune-modulating agent may allow fora lower dose of the BO-11X formulation and/or one or moreimmune-modulating agent (e.g. as compared to monotherapy) and therebyincrease the therapeutic window of either agent. In some embodiments,the lowered dose mitigates one or more side effects without (or minimal)loss of efficacy. In some embodiments, the BO-11X formulation and/orimmune-modulating agent is used to treat a subject that has atreatment-refractory cancer. In some embodiments, the BO-11X formulationis used to treat a subject that is refractory to one or more immune-modulating agents, in particular the one that is actually combined withthe BO-11X formulation.

For instance, in some embodiments, the subject is refractory to a PD-1and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO), pembrolizumab (KEYTRUDA),pidilizumab (CT-011), MK-3475, BMS-936559, Ibrutinib, and/orMPDL328OA-refractory patients. For instance, in some embodiments, thesubject is refractory to an anti-CTLA-4 agent, e.g. ipilimumab(Yervoy)-refractory patients (e.g. melanoma patients). In someembodiments, the subject is refractory to a BO-11X formulation.Accordingly, in various embodiments the present invention providesmethods of cancer treatment that rescue patients that are non-responsiveto various therapies, including monotherapy of an BO-11X formulation orone or more immune-modulating agents.

In various embodiments, the terms “patient” and “subject” are usedinterchangeably. In some embodiments, the subject and/or animal is amammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, rabbit, sheep, or non-human primate, such as a monkey (e.g. baboon)or chimpanzee.

In various embodiments, methods of the invention are useful in treatmenta human subject. In some embodiments, the human is a pediatric human. Inother embodiments, the human is an adult human. In other embodiments,the human is a geriatric human. In other embodiments, the human may bereferred to as a patient or a subject. In some embodiments, the human isa female. In some embodiments, the human is a male.

Treatment Regimens and Combination Therapies

In some embodiments, present invention provides for specific cancertreatment regimens with BO-11X formulations and immune-modulating agents(and optionally one or more additional therapeutic agent). For example,in some embodiments, the BO-11X formulation, e.g. BO-111 OR BO-112, isadministered to a patient first to normalize tumor vascularization,optionally by reducing or ablating hypoxia. Such first administration ofthe BO-11X formulation, e.g. BO-111 OR BO-112, may stimulate and/orincrease T lymphocytes (e.g. CD4+and CD8+T cells) and/or NK cells tumorand/or inhibit and/or decrease recruitment of immunosuppressive cells(e.g. myeloid-derived suppressor cells (MDSCs), regulatory T cells(Tregs); tumor associated neutrophils (TANs), M2 macrophages, and tumorassociated macrophages (TAMs)) to the tumor. In some embodiments, thepresent therapies may alter the ratio of M1 versus M2 macrophages in thetumor site to favor M1 macrophages. Notably, unlike for example,anti-angiogenic molecules, the BO-11X formulations, in some embodiments,induce a long lasting

(i.e. greater than transient) vascular normalization. For example,BO-11X formulation-vascular normalization may last greater than 1, or 2,or 3, or 4, or 5, or, or 6, or 7, or 14 days, or 21 days. Accordingly,in some embodiments, this long-lasting BO-11X formulation-vascularnormalization allows for a sustainable permissive tumor microenvironmentthat is more likely to be responsive to one or more immune-modulatingagents. That is, in some embodiments, the BO-11X formulation potentiatesimmune-modulating agent therapy.

Alternatively, the BO-11X formulation, e.g. BO-111 OR BO-112, isadministered to a patient after treatment with one or moreimmune-modulating agents. For instance, in some embodiments, theimmune-modulatory agent targets one or more co-inhibitory molecules andreduces or eliminates immunosuppression. In this favorable context, i.e.upon removal of suppression, the BO-11X formulation, e.g. BO-111 ORBO-112, is administered is administered to stimulate the immune system.Or the immune-modulatory agent targets one or more co-stimulatorymolecules first and the BO-11X formulation, e.g. BO-111 OR BO-112, isadministered is administered second to bolster this effect, for example,synergistically.

Further, as described herein, the BO-11X formulation and/orimmune-modulating agent can be combined with an additional therapeuticagent in the context of, for example, co-administration, a treatmentregimen or a co-formulation.

In some embodiments, the BO-11X formulation and/or immune-modulatingagent, optionally with an additional therapeutic agent, can beadministered sequentially. The term “sequentially” as used herein meansthat the additional therapeutic agent and the BO-11X formulation and/orimmune-modulating agent are administered with a time separation of morethan about 60 minutes. For example, the time between the sequentialadministration of the additional therapeutic agent and the BO-11Xformulation and/or immune-modulating agent can be more than about 60minutes, more than about 2 hours, more than about 5 hours, more thanabout 10 hours, more than about 1 day, more than about 2 days, more thanabout 3 days, or more than about 1 week apart. The optimaladministration times may depend on the rates of metabolism, excretion,and/or the pharmacodynamic activity of the additional therapeutic agentand the BO-11X formulation and/or immune-modulating agent beingadministered. Either the additional therapeutic agent or the presentagents may be administered first.

In some embodiments, the BO-11X formulation and/or immune-modulatingagent, optionally with an additional therapeutic agent, can beadministered simultaneously. The term “simultaneously” as used herein,means that the additional therapeutic agent and the BO-11X formulationand/or immune-modulating agent are administered with a time separationof no more than about 60 minutes, such as no more than about 30 minutes,no more than about 20 minutes, no more than about 10 minutes, no morethan about 5 minutes, or no more than about 1 minute. Administration ofthe additional therapeutic agent and BO-11X formulation and/orimmune-modulating agent can be by simultaneous administration of asingle formulation (e.g. a formulation comprising the additionaltherapeutic agent and the BO-11X formulation and/or immune-modulatingagent) or of separate formulations (e.g., a first formulation includingthe additional therapeutic agent and a second formulation including theBO-11X formulation and/or immune-modulating agent).

Co-administration also does not require the additional therapeuticagents to be administered to the subject by the same route ofadministration. Rather, each therapeutic agent can be administered byany appropriate route, for example, parenterally or non-parenterally.

Such a combination may lead to synergism and/or additive and/or potenteffects at a lower dose of the BO-11X formulation and/orimmune-modulating agent. For example, when the BO-11X formulation iscombined with one or more immune-modulating agents the effective amountof the BO-11X formulation may be lower than what it would be in amonotherapy. In some embodiments, the BO-11X formulation is combinedwith an immune-modulating agent and the effective amount of the BO-11Xformulation is a sub-therapeutic dose, for example, when theimmune-modulating agent is combined with a BO-11X formulation theeffective amount of the immune-modulating agent may be lower than whatit would be in a monotherapy. In some embodiments, the immune-modulatingagent is combined with a BO-11X formulation and the effective amount ofthe immune-modulating agent is a sub-therapeutic dose. In variousembodiments, the immune- modulating agent is combined with a BO-11Xformulation and an additional therapeutic agent and the effective amountof the additional therapeutic agent is a sub-therapeutic dose. The term“sub-therapeutic dose or amount” means that a dose or amount of apharmacologically active substance is below the dose or amount of thatsubstance that is administered, as the sole substance, to achieve atherapeutic effect. The sub-therapeutic dose of such a substance mayvary depending upon the subject and disease condition being treated, theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. In one embodiment, thesub-therapeutic dose or amount of the chemotherapeutic agent is lessthan 90% of the approved full dose of the chemotherapeutic agent, suchas that provided in the U.S. Food & Drug Administration-approved labelinformation for the chemotherapeutic agent. In other embodiments, thesub- therapeutic dose or amount of the chemotherapeutic agent is lessthan 80%, 70%, 60%, 50%, 40%, 30%, 20% or even 10% of the approved fulldose, such as from 20% to 90%, 30% to 80%, 40% to 70% or another rangewithin the values provided herein.

In some embodiments, the effective amount of the immune-modulating agentis less than an effective amount used in monotherapy for the same cancerand/or a combination therapy with an agent besides a BO-11X formulationfor the same cancer. In some embodiments, the effective amount of theBO-11X formulation is less than an effective amount used in monotherapyfor the same cancer or clinical status, and/or a combination therapywith an agent (such as an immune-modulating agent) for the same canceror clinical status.

In various embodiments, the BO-11X formulation is combined with one ormore immune-modulating agents (e.g. 1, or 2, or 3, or 4, or 5immune-modulating agents) and, optionally, one or more additionaltherapeutic agents (e.g. 1, or 2, or 3, or 4, or 5 additionaltherapeutic agents). Such combinations may lead to synergism and/oradditive and/or potent effects at a lower dose of the BO-11X formulationand/or immune-modulating agent and/or the one or more additionaltherapeutic agents. Co-administration may be simultaneous or sequential.Further the pharmaceutical compositions including the BO-11X formulationand/or immune-modulating agent may comprise the additional therapeuticagent (e.g. via co-formulation). That is, in some embodiments, two ormore of any of the agents disclosed herein may be co-formulated.Further, in some embodiments, the BO-11X formulation and/orimmune-modulating agent may be administered to a patient that isundergoing treatment with one or more additional therapeutic agent.Further, in some embodiments, the BO-11X formulation and/orimmune-modulating agent may supplant a patient's current treatment withone or more additional therapeutic agent.

Adjuvant therapy, also called adjuvant care, is treatment that is givenin addition to the primary, main or initial treatment. By way ofnon-limiting example, adjuvant therapy may be an additional treatmentusually given after surgery where all detectable disease has beenremoved, but where there remains a statistical risk of relapse due tooccult disease. In some embodiments, the agents described herein areused as an adjuvant therapy in the treatment of a cancer. In someembodiments the therapeutic agents described herein are administered asa neo-adjuvant therapy prior to resection. In certain embodiments,neo-adjuvant therapy refers to therapy to shrink and/or downgrade thetumor prior to any surgery. In some embodiments, neo-adjuvant therapymeans a therapeutic agent described herein is administered to cancerpatients prior to surgery or other technique allowing tumor ablation.

In some embodiments the therapeutic agents described herein are usefulas a maintenance therapy after an initial treatment with a first-linetherapy, including without limitation any of the additional therapeuticagents of the present disclosure.

In various embodiments, the present invention provides a treatmentregimen or a method for treating cancer or tumors in a subject thatincludes administering simultaneously or sequentially a therapeuticallyeffective amount of a BO-11X formulation and/or an immune-modulatingagent and one or more of the additional therapeutic agents describedherein. In various embodiments, the present invention provides atreatment regimen or a method for treating cancer or tumors in a subjectthat includes administering simultaneously or sequentially atherapeutically effective amount of a BO-11X formulation and/or animmune-modulating agent and one or more of the anti-cancer agentsdescribed herein, including but not limited to chemotherapeutic agents.Suitable chemotherapeutic agents to be used in the methods of thepresent invention may include those described herein. In certainembodiments, the chemotherapeutic agent is one or more of 5-fluorouracil(5-FU), doxorubicin, gemcitabine, paclitaxel, and cisplatin. By way ofexample, in some embodiments, the present invention provides combining aBO-11X formulation and/or an immune-modulating agent with one or morecommon cancer treatment regimens (by way of non-limiting illustration,FOLFOX, FOLFIRI, IFL, FL (Mayo), QUASAR, Machover schedule, CAF, CMF,ECF, and FEC).

In various embodiments, the additional therapeutic agent is anantihyperproliferative agent. Antihyperproliferative agents include, butare not limited to, doxorubicin, daunorubicin, mitomycin, actinomycin D,bleomycin, cisplatin, VP16, enedyine, taxol, vincristine, vinblastine,carmustine, melphalan, cyclophsophamide, chlorambucil, busulfan,lomustine, 5-fluorouracil, gemcitabin, BCNU, or camptothecin.

In addition, the additional therapeutic agent can further include theuse of radiation. In addition, the methods of treatment can furtherinclude the use of photodynamic therapy.

Salts, Pharmaceutical Compositions and Doses

In some embodiments, the present invention provides for the agentsdescribed herein and pharmaceutically acceptable esters, pro-drugs,salts, solvates, enantiomers, stereoisomers, active metabolites,co-crystals, and other physiologically functional derivatives thereof.

In one aspect, the present invention provides agents described herein,and a pharmaceutically acceptable carrier or excipient. Thepharmaceutical composition can be in any suitable form appropriate forthe desired use and route of administration. Pharmaceutical excipientscan be liquids, such as water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The pharmaceutical excipients canbe, for example, saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea and the like. In one embodiment, thepharmaceutically acceptable excipients are sterile when administered toa subject. Water is a useful excipient when any agent described hereinis administered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid excipients,specifically for injectable solutions. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmono-stearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Other examples ofsuitable pharmaceutical excipients are described in Remington'sPharmaceutical Sciences (edited by Allen, Loyd V., Jr; 22^(nd) edition,2012).

Additionally, the pharmaceutical compositions of the present inventionmay contain adjuvants such as preservatives, wetting agents, emulsifyingagents, pH buffering agents, and dispersing agents. Further, auxiliary,stabilizing, thickening, lubricating, and coloring agents can beincluded. Prevention of the action of microorganisms may be ensured bythe inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Thepharmaceutical compositions may also include isotonic agents such assugars, sodium chloride, and the like.

Where necessary, the pharmaceutical compositions can also include asolubilizing agent. Also, the agents can be delivered with a suitablevehicle or delivery device as known in the art. Compositions foradministration can optionally include a local anesthetic such as, forexample, lidocaine to lessen pain at the site of the injection.

The pharmaceutical compositions of the present invention can take theform of solutions, suspensions, emulsions, drops, tablets, pills,pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. Thus, thecomposition described herein may be comprised in a capsule, tablet,pill, caplet, bottle, ampoule, sachet, syringe, cartridge, nebulizer orother container. In one embodiment, the pharmaceutical composition is inthe form of a capsule. In another embodiment, the pharmaceuticalcomposition is in the form of a tablet.

In some embodiments, the administration of any of the described agentsand compositions is any one of oral, intravenous, and parenteral. Invarious embodiments, routes of administration include, for example:oral, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, sublingual, intranasal,intracerebral, intrahepatic, intrapancreatic, intravesicular,intravaginal, transdermal, rectally, by inhalation, or topically, forexample, to the ears, nose, eyes, or skin. In some embodiments, theadministering is effected orally or by parenteral injection. The mode ofadministration can be left to the discretion of the practitioner, anddepends in part upon the site of the medical condition and/or concurrenttreatments (being, for instance, chemotherapy, radiotherapy, or incombination with antibodies, vaccines and other cancer-targeting drugs).In various embodiments, administration results in the release of anyagent described herein into the bloodstream.

Any agent and/or pharmaceutical composition described herein can beadministered orally. Such agents and/or pharmaceutical compositions canalso be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or muco-cutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anadditional therapeutic agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used. Inspecific embodiments, it may be desirable to administer locally to thearea in need of treatment.

In one embodiment, an agent described herein and/or pharmaceuticalcomposition described herein is formulated in accordance with routineprocedures as a composition adapted for oral administration to humans.Solid dosage forms for oral administration include, for example,capsules, tablets, pills, powders, and granules. In such dosage forms,the active agent is mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier such as sodium citrate, di-calciumphosphate, etc., and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, silicic acid, microcrystallinecellulose, and Bakers Special Sugar, etc., b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinyl-pyrrolidone, etc., c) humectants such as glycerol, etc., d)disintegrating agents such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, sodium carbonate,cross-linked polymers such as crospovidone (cross-linkedpolyvinylpyrrolidone), croscarmellose sodium (cross-linked sodiumcarboxymethylcellulose), sodium starch glycolate, etc., e) solutionretarding agents such as paraffin, etc., f) absorption accelerators suchas quaternary ammonium agents, etc., g) wetting agents such as, forexample, cetyl alcohol and glycerol monostearate, etc., h) absorbentssuch as kaolin and bentonite clay, etc., and i) lubricants such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, glycerylbehenate, etc., and mixtures of such excipients.One of skill in the art will recognize that particular excipients mayhave two or more functions in the oral dosage form. In the case of anoral dosage form, for example, a capsule or a tablet, the dosage formmay also comprise buffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active agents, the liquid dosage forms may contain inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ,olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Dosage forms suitable for parenteral administration (e.g.intravenous, intramuscular, intrahepatic, intrapancreatic,intraperitoneal, subcutaneous and intra-articular injection andinfusion) include, for example, solutions, suspensions, dispersions,emulsions, and the like. They may also be manufactured in the form ofsterile solid compositions (e.g. lyophilized composition), which can bedissolved or suspended in sterile injectable medium immediately beforeuse. They may contain, for example, suspending or dispersing agentsknown in the art. Pharmaceutical compositions of this invention forparenteral injection comprise pharmaceutically acceptable sterileaqueous or non-aqueous solutions, dispersions, suspensions or emulsionsas well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use. Examples of suitable aqueousand non-aqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such as olive oil) and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Any agent described herein and/or pharmaceutical composition describedherein can be administered by controlled-release or sustained-releasemeans or by delivery devices that are known to those of ordinary skillin the art. Such dosage forms can be useful for providing controlled- orsustained-release of one or more active ingredients using, for example,hydropropyl cellulose, hydropropylmethyl cellulose,polyvinylpyrrolidone, Eudragit, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled- orsustained-release formulations can be readily selected for use with theactive ingredients of the agents described herein. The invention thusprovides single unit dosage forms suitable for oral administration suchas, but not limited to, tablets, capsules, gelcaps, and caplets that areadapted for controlled- or sustained-release.

Formulations comprising the agents described herein and/orpharmaceutical compositions of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodsknown in the art of pharmacy. Such methods generally include the step ofbringing the therapeutic agents into association with a carrier, whichconstitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g. wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art).

It will be appreciated that the actual dose of the agents describedherein and/or pharmaceutical compositions of the present invention to beadministered according to the present invention may vary according tothe particular agent, the particular dosage form, and the mode ofadministration. Many factors that may modify the action of the BO-11Xformulations (e.g. body weight, gender, diet, time of administration,route of administration, rate of excretion, condition of the subject,drug combinations, genetic disposition and reaction sensitivities) canbe taken into account by those skilled in the art. Administration can becarried out continuously or in one or more discrete doses within themaximum tolerated dose. Optimal administration rates for a given set ofconditions can be ascertained by those skilled in the art usingconventional dosage administration tests.

Individual doses of the agents described herein and/or pharmaceuticalcompositions of the present invention can be administered in unit dosageforms (e.g., tablets or capsules) containing, for example, from about0.01 mg to about 1,000 mg of poly(I:C) molecules within the BO-11Xformulation [e.g. wherein said individual BO-11X formulation is formedby making a complex from about 0.01 mg to about 1000 mg of poly(I:C)],inclusive of all values and ranges there between. In some embodiments,the agents described herein and/or pharmaceutical compositions of thepresent invention are administered at an amount of from about 0.01 mg toabout 1000 mg of poly(I:C) molecules within the BO-11X formulationdaily, or from about 0.1 mg to about 10 mg daily [e.g. wherein saidindividual daily BO-11X formulation is formed by making a complex fromabout 0.01 mg to about 1000 mg, preferably about 0.01 mg to 10 mg ofpoly(I:C)], inclusive of all values and ranges there between.

In some embodiments, a suitable dosage of the agents and/orpharmaceutical compositions of the present invention is in a range ofabout 0.001 to about 10 mg of poly(I:C) molecules within the BO-11Xformulation/kg of body weight of the subject, preferably 0.003 to about10 mg of poly(I:C) molecules within the BO-11X formulation/kg of bodyweight of the subject, more preferably 0.005 to about 10 mg of poly(I:C)molecules within the BO-11X formulation/kg of body weight of the subjectand even more preferably 0.01 to about 10 mg of poly(I:C) moleculeswithin the BO-11X formulation per kg of body weight of the subject [e.g.wherein said individual BO-11X formulation administered is formed bymaking a complex from about 0.005 mg to about 10 mg of poly(I:C)] per kgof body weight of the subject, preferably about 0.003 mg to about 10 mgof poly(I:C)] per kg of body weight of the subject, more preferablyabout 0.001 mg to about 10 mg of poly(I:C)] per kg of body weight of thesubject, inclusive of all values and ranges there between. In otherembodiments, a suitable dosage of the BO-11X formulation and/orimmune-modulating agent and/or additional therapeutic agent is in arange of about 0.01 mg/kg to about 10 mg/kg of body weight, or in arange of about 0.05 mg/kg to about 1 mg/kg of body weight.

In accordance with certain embodiments of the invention, the agentsand/or pharmaceutical compositions described herein may be administered,for example, more than once daily, about once per day, about every otherday, about every third day, about once a week, about once every twoweeks, about once every month, about once every two months, about onceevery three months, about once every six months, or about once everyyear.

Kits

The invention also provides kits that can simplify the administration ofthe agents and/or pharmaceutical compositions described herein. The kitis an assemblage of materials or components, including at least one ofthe agents described herein. The exact nature of the componentsconfigured in the kit depends on its intended purpose. In oneembodiment, the kit is configured for the purpose of treating humansubjects.

Instructions for use may be included in the kit. Instructions for usetypically include a tangible expression describing the technique to beemployed in using the components of the kit to affect a desired outcome,such as to treat, for example, cancer, diabetes, or obesity. Optionally,the kit also contains other useful components, such as, diluents,buffers, pharmaceutically acceptable carriers, syringes, catheters,applicators, filters, (micro)needles, pipetting or measuring tools,bandaging materials or other useful paraphernalia as may be readilyrecognized by those of skill in the art.

The materials and components assembled in the kit can be provided to thepractitioner store in any convenience and suitable ways that preservetheir operability and utility. For example, the components can beprovided at room, refrigerated or frozen temperatures. The componentsare typically contained in suitable packaging materials. In variousembodiments, the packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging material may have an external label which indicates thecontents and/or purpose of the kit and/or its components.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

EXAMPLES Example 1: Effects of Complex-Size on Anticancer Effects ofDistinct JetPEI-Based Poly(I:C) Preparations (BO-111 Formulations)Materials & Methods BO-111 Formulations (2-Vial Process)

The single-stranded polyinosinic acid [poly(I)] and polycytidylic acid[poly(C)] molecules that were used for generating double-strandedpolyinosinic-polycytidylic acid [poly(I:C)] molecules] were obtainedfrom commercial providers such as Tide Group, Carbogen or Invivogen.Depending from the provider and the batch, the size distribution forpoly(C) molecules is defined as being: <400 bases, 20-82% (with furthertests performed using preparations presenting, for instance, 33%, 43%,or 50%); 400-850 bases, 15-40% (with further tests performed usingpreparations presenting, for instance, 27%, 30% or 37%); 850-5000 bases,3-50% (with further tests performed using preparations presenting, forinstance, 13%, 30% or 34%); >5000 bases, 1% or less (generally absent).Depending from the provider and the batch, the size distribution forpoly(I) molecules is defined as being: <400 bases, 80-95% (with furthertests performed using preparations presenting, for instance, 86% and91%); 400-850 bases, 5-20% (with further tests performed usingpreparations presenting, for instance, 8% or 12%); 850-5000 bases, 0˜5%(with further tests performed using preparations presenting, forinstance, 1% or below); >5000 bases, 1% or less (generally absent).Acceptance criteria for manufacturing BO-111 formulations that apply topoly(I) and poly(C) powder or solutions also include maximum absorption(at wavelength of 248±1 nm and of 268±1 nm for poly(I) and poly(C),respectively), endotoxin content (≤10 EU/mg), pH (6.0-8.0), andsedimentation coefficient (≥4S).

Batch poly(I) preparations were obtained by dissolving powder poly(I)(1.0 eq., 23.99 g) at 50° C. in PBS 1×(2.4 L) with continuous stirringinto a 6-liter flask. Batch poly(C) preparations were obtainedseparately using powder poly(C) in the same manner and at the sameconcentration. Additional steps of filtration could be implemented tofurther improve quality of starting solutions using membranes with a 300kDa cut-off or a 500 kDa cut-off (Pellicon 2 cassette, Millipore). Thepermeates of these filtration steps are concentrated and freed fromsmall size impurities, such as monomers, over a 30 kDa membrane(Pellicon 2 cassette, Millipore). The resulting retentate for eachsolution is mixed with a concentrated buffer solution (such as PBS 10X).For both solutions, the optical density was determined to calculate theconcentration as a basis for a 1:1 stoichiometry for the followingannealing step, adjusting consequently the total volume before annealingstep. Poly(I) solution is mixed and stirred with poly(C) solutionmolecules at 55-62° C. for 30 minutes. The resulting solution is slowlycooled down at room temperature for approx. 3 hours for annealingsingle-stranded molecules and generating poly(I:C) molecules, andfinally filtered over a G3 glass pore filter (pore size of approx. 15-40μm).

This annealing process generates a solution containing a pool ofdifferent double-stranded poly(I:C) molecules that is then applied on achromatographic GPC column. The chromatography is performed with Omnifitglass column of 5 cm diameter that was filled with a slurry of 700 mLToyopearl HW-65F in 40 mM sodium phosphate buffer. The slurry wasallowed to settle slowly, followed by washing with 40 mM sodiumphosphate buffer (pH=6.9) at increasing flow rate from 10 mL/min to 60mL/min. The column was installed in a preparative HPLC device consistingof two feed pumps, a UV detector, sampling valves and a computer. Thereaction mixture from the annealing was loaded on the column and elutedwith 40 mM sodium phosphate buffer (flow=50 mL/min, pH=6.9). Targetfractions were taken when the UV signal was between 100 mV and 1250 mV,and pooled for further work-up using four desalting cycles of dilutionand concentration using a tangential flow device (TFF, MilliporePellicon 2, regenerated cellulose, equipped with three membranecassettes of 0.1 m² each, 300 kDa cut-off). Inlet and outlet wereconnected to the first glass bottle with the pooled chromatographyfractions. Final retentate and washing solution were filtered over amembrane to give a clear, colorless solution that was desalted usingisopropanol, freeze-dried, and lyophilized at room temperature (at 1mbar for approx. 5 days).

Different commercial In vivo-JetPEI [having an average molecular weightcomprised between 8.3 and 22.5 kDa, and a polydispersity index <1.5, asdetermined from that of PEOX (polyethyleneoxide, precursor to PEI) byGel Permeation Chromatography (GPC: SOP GPC-0044) and sterile filteredthrough a 0.2 μm filter] was obtained from PolyPlus (catalog no.201-50G). The 2-vial process involves mixing the content of a Vial 1containing poly(I:C) molecules (volume of 1.0 mL or less, when fastpipetting the solutions, or up to 5.5 mL, when using a syringe) with aVial 2 containing PEI solution (volume of 1.0 or less mL, when fastpipetting the solutions, or up to 5.5 mL, when using a syringe).Alternatively, the content of Vial 1 is aspirated with a syringe (10 mL)and needle (G20-0.9 μm) and quickly shot over the surface of the liquidin Vial 2. Resulting BO-111 preparation is then filtered through amembrane having a pore size in the 1-5 μm range, ensuring elimination oflarger, visible particles. Glucose (or mannitol) was included as anexcipient in Vial 1 to reach 5% (w/v) concentration in the final BO-111preparation [i.e. said composition is formed by additionally addingglucose (or mannitol) in a concentration of 5% (weight/total volume ofsaid composition)]. Glucose has been extensively used as an excipientthat promotes an acceptable osmolality of BO-111 (302 mOsm/Kg) withoutcompromising functional or physico-chemical features and avoidingpotential undesired side effects due to the administration of mannitolat high concentrations.

Distinct BO-111 preparations were produced by filtering once or twicethe initial BO-111 solution through cellulose acetate membrane ofdifferent pore size (Minisart® NML Syringe Filters; Sartorius) accordingto manufacturer's instructions. Alternatively, the initial BO-111preparation was centrifuged for 15 minutes at the indicated speed usinga fixed-angle rotor FA-45-24-11 for Centrifuges 5415D/5415R (Eppendorf).The flow through of membrane filtration and the centrifugationsupernatant, respectively, were stored until use at 4° C. at a poly(I:C)concentration of 0.5-0.8 mg/mL as determined by UV. Poly(I:C)concentration is then re-calculated before each experiment, generatingsample with the same dose for every condition.

The size of poly(I:C) molecules within BO-111 preparations wasdetermined using agarose gels and unlabeled or [³²P] labeled poly(I) andpoly(I:C) preparations. Briefly, 1 μg of poly(I) and poly(I:C) (PBS) areloaded into the agarose gel and electrophoresis was performed for 1 hourat 80 volt in TBE buffer. Depending from the size distribution ofinitial poly(C) and poly (I) molecules, the size distribution ofpoly(I:C) molecules that are present in BO-111 preparations wasdetermined as being: <400 bases, 7-57% (with further tests performedusing preparations presenting, for instance, 15% or 21%); 400-850 bases,20-45% (with further tests performed using preparations presenting, forinstance, 25% or 27%); 850-5000 bases, 20˜70% (with further testsperformed using preparations presenting, for instance, 52% or53%); >5000 bases, 0-9% (with further tests performed using preparationspresenting, for instance, 1% or 0%).

Analytical Technologies

The value for zeta average (z-average) diameter and polydispersity indexof JetPEI/poly(I:C) particles in distinct BO-111 preparations (between0.5-0.8 mg/mL, to be diluted for cell-based and other assays at apoly(I:C) concentration of 1.0 μg/mL) were determined using ZetasizerNano ZS according to the manufacturer's instructions and in accordancewith ISO 22412, based on the assumption that said particles arespherical. In general, dynamic light scattering (Nanosizer technology)is applied using v7.11 software.

Functional Characterization of BO-111 Preparations

The different BO-111 preparations were tested using human melanomacells, human pancreatic cells, or human melanocytes according to theliterature describing the properties of BO-110 complexes (Pozuelo-RubioM et al., 2014; Tormo D et al., 2009; WO2011003883). Briefly, cellviability assays were performed on adherent cells at least 12 hoursbefore treatment. The percentage of cell death at the indicated timesand treatment concentrations was estimated by standard trypan blueexclusion assays on floating and adherent cells that were pooled,stained with a 0.4% trypan blue solution (Gibco Laboratories, GrandIsland, N.Y., USA) and scored under a light microscope (a minimum of100-500 cells per treatment were counted). Each preparation was testedfor a period comprised between 12 hours and 48 hours and atconcentrations poly(I:C) molecules in the different preparations thatwere comprised between 0.1 and 2.5 μg/mL.

Results

Existing process for the preparation of BO-110 complexes as described inthe literature (Pozuelo-Rubio Met al., 2014; Tormo D et al., 2009;WO2011003883) have been established on a laboratory scale and have someimportant limitations with respect to requirements for generatingmaterials that can be tested on larger pre-clinical scale, and then forclinical evaluation: limited concentration (not beyond 0.05 mg/mL) andlimited possibility for up-scaling and, at the same time, making themanufacturing process GMP-compliant. In particular, the manufacturingprocess should allow producing batches of formulation comprisingpoly(I:C)-containing particles having physico-chemical features (such assterility, particle size distribution, stability, lack of visible andsub-visible particles, and concentration of at least 0.5 mg/mL) asuniform as possible among distinct preparations for correctly evaluatingtheir biological effects and medical use in relevant pre-clinical modelsand pharmaco-toxicological assays.

A first step in reaching these objectives was to substitute the step ofadding drop-by-drop poly(I:C) solution to JetPEI solution (or other wayaround, as in initial BO-110 preparations; WO2011003883) and incubatingthe mixture at room temperature. The speed of mixing was identified as apotentially important, yet not evaluated, factor for solving themanufacturing problem. At this scope, a new type of poly(I:C)-PEIformulation, named as BO-111, was established by substituting theapproach of bulk mixing of solutions to be lyophilized and packaged intovials, with the approach of producing two vials, each containing thedesired amount of JetPEI and poly(I:C) molecules. The content of the twovials are rapidly mixed by injecting (or by other means for rapidlymixing liquids, such as fast pipetting), the two solutions havingsimilar volume. The resulting solution has a volume compatible withlater assays and uses (e.g. a 1.2 mL BO-111 preparation at 0.5 mg/mL,resulting from mixing 2 solutions, each having a volume of 0.6 mL).

The syringe and needle used for the mixture generate enough turbulencefor promoting fast mixture and rapid formation of particles in BO-111preparations, with limited (or absent) visible particulate. Specifictechnical details of BO-111 manufacturing process may be adapted inorder to provide a further increased level of activity, reproducibility,stability, and/or homogeneity of BO-111 preparations, for instance byextracting salts, eliminating production residues, filtering thesolutions with filters with large pore sizes (e.g. in the range between1 and 5 μm), fast pipetting or vortexing the two solutions, selectingsyringe size/diameter, quickly adding poly(I:C) solution to JetPEIsolution and not the opposite, or lyophilizing preparations usingcompounds like glucose or mannitol as excipient.

However, such details can hardly be transferred from small volumes forsingle or immediate use to a larger scale preparations of GMP-compliant,pharmaceutical formulations that are based on JetPEI as carrier andcontain poly(I:C) molecules at the sufficiently high concentration (atleast 0.5 mg/mL) and uniform concentrations that are required fortesting high doses during pharmaco-toxicological or other pre-clinicalevaluation. Moreover, the step of mixing components of BO-111 justbefore administration leaves in the technician's hands the good qualityof the final material, in particular with respect to formation of clear,not turbid solutions that contain BO-111 particles having larger, andnot fully controlled, diameter (size) range.

In this context, the aim in the next step was to evaluate differentmeans and effects of standardizing the diameter of BO-111 particlesmixture. The initial BO-111 preparation that was obtained by the 2-vial,fast pipetting process was used for generating and comparing alternativepreparations in which concentration and/or size of the solute, such asBO-111 complexes, are altered within the solution by using commontechnologies such as centrifugation at high speed (e.g. beyond 5000 rpm)and filtration (e.g. with pore size in the 1-5 μm range or in asub-micromolar range).

This initial analysis shows that, by applying either technologies, theresulting BO-111 preparations not only presents a reduction in averageBO-111 particle diameter, but also a surprisingly increase ofcytotoxicity of such preparations against cancer cells that isproportional to the decreased average diameter of BO-111 complexes (FIG.1A). Such increased anticancer effect was also confirmed in a doseresponse study showing that the flow-through BO-111 solution, beingobtained by filtering the initial BO-111 solution with the largersub-micromolar pore size, presents an already important increase of theanticancer effects of BO-111 complexes, especially at lower BO-111concentrations (FIG. 1B). A similar increase in cytotoxicity is alsoconfirmed by using BO-110 preparations that are filtered through afilter having a 0.8 μm pore size.

Further criteria that can be evaluated are those related to JetPEIfeatures and ratio with respect to poly(I:C) molecules. At thestructural level, JetPEI preparations including linear PEI within arange of average molecular weight were tested using the 2-vial process.The comparison of the cytotoxicity of such BO-111 preparations (FIG. 2A)showed that linear PEI of higher molecular weight (e.g. between 17 and23 kDa) provide anticancer BO-111 preparations that are more effectivethan those including linear PEI having lower molecular weight (e.g. 8.3kDa).

In addition to defining a range of linear PEI sizes that are suitablefor manufacturing BO-111 having the desired anticancer properties, theeffect of different concentration ratio between amines of PEI andanionic phosphate of poly(I:C) molecules was tested in the context ofBO-111 manufacturing. The ionic balance between JetPEI and poly(I:C)molecules may provide complexes that present different level ofinteractions with cellular components (e.g. for effective cell entry).This balance is calculated as the N/P ratio, defining the number ofnitrogen residues of JetPEI per polyribonucleotide phosphate, a valuethat, for in vivo polyribonucleotide delivery experiments, isrecommended as being between 6 and 8 (avoiding the toxicity problemsbeyond 8, that is 0.16 μL JetPEI per μg double-stranded poly(I:C)molecules). Distinct BO-111 preparations showed a dose-responsecytotoxic effect on both melanoma cells and normal melanocytes whenincreasing such ratio N/P from 1.8 to 5.2. Indeed, only at anintermediate range (around the ratio N/P of 3) cytotoxicity againstmelanoma cells is well superior to the one against normal melanocytes,viability of latter cells being only marginally affected by thisspecific BO-111 preparation when compared to untreated cells (FIG. 2B).

The BO-111 preparations have also been analyzed for the content ofpoly(I:C) molecules using labeled or unlabeled poly(I:C) batches. Theinitial poly(I:C) batch preparation comprises double stranded poly(I:C)molecules that, consequently to their manufacturing and their annealing,have a size distribution up to 5 kilobases of length (or more), with atleast 40% or 50% of such double-stranded with a size higher than 0.85 Kband at least 70% of such double-stranded polyribonucleotides with a sizecomprised between 0.4 and 5 Kb (in a representative preparation, <400basepairs (bp): 21%, 400-850 bp: 27%, 850-5000 bp 52%). When poly(I:C)molecules are associated to JetPEI in complexes within BO-111preparations using the 2-vial process, the totality of poly(I:C)molecules is associated with JetPEI, as shown by agarose gel analysiswith unlabeled or labeled poly(I:C) preparations (FIG. 3A and 3B). Byelectrophoresis it has also been determined that, in BO-111 preparationsat N/P ratio below 3, poly(I:C) molecules are not fully associated withJetPEI, as it can be observed with higher N/P ratio (3 or above; FIG.3C). Thus, an appropriate manufacturing process allows incorporatingefficiently poly(I:C) molecules with a wide size distribution intobiologically functional BO-111 preparations, without adding a specificprocedure for removing uncomplexed poly(I:C) molecules or JetPEI.

Structural BO-111 features were evaluated also by using technologies forestablishing the size distribution of particles in the sub-micrometerrange when the manufacturing process is repeated or modified, or whenBO-111 preparations are tested for their stability. The reproducibilityof this manufacturing process is demonstrated given that distinct BO-111preparations are always presenting a mono-modal distribution of particlesize, with diameters that are mostly concentrated around 100 nm, mostoften between 50 and 90 nm (average diameter (d· nm) of 85-90 nm), witha large majority of particles having a size below 100 nm, or 200 nm, butnot exceeding 400 nm, but commonly even not exceeding 300 nm (FIG. 4A).When this reference BO-111 preparation is exposed to variations intemperature or incubation time, the diameter distribution can change,but it is still mono-modal, peaking between 100 and 150 nm (averagediameter (d·nm) of 105-110 nm), with a large majority of particles stillhaving a diameter below 300 nm and not exceeding 600 nm (FIG. 4B and4C). The corresponding BO-111 preparations still present a similarcytotoxicity level when tested as described for FIGS. 1 and 2,demonstrating that this mixing procedure can provide formulationsconsistent with requirements for pharmaceutical development. However,modifications of the process, such as reducing the mixing speed or notintroducing filtering procedures may alter the size distribution ofBO-111 complexes, becoming bi-modal, with a poorly controlled and higheraverage size (d·nm) and a large majority of particles exceeding 500 nm(FIG. 4D).

These experiments show that the manufacturing and formulation process ofthe complex named BO-110 can be improved by applying a fast andcontrolled mixing of components at small scale prior to their use,leading to a more uniform, concentrated, and effective poly(I:C)-basedpreparation named as BO-111. However, further improvements are needed togenerate preparations presenting fully GMP-compliant preparations forthe highest stability and efficacy features independently from aprocedure to be established just before medical use. In particular, the“fast pipetting” method or other variations of the 2-vial process maystill provide a considerable amount of particles having a diameter above200 nm (up to 1 μm or several micrometres). The additional (and possiblyonly partially efficient) filtration step that would be required inorder to provide the desired sterile and concentrated BO-111preparations (presenting functional complexes of smaller, more narrowlydistributed, uniform diameter around 100 nm) can have the consequence oflosing a large amount of material that is retained in the membranefilter. Thus, even though the initial approach has allowed establishingsmall scale BO-111 preparations with an inverse relationship betweenBO-111 complex diameter and its therapeutic activity, further technicalimprovements are required for establishing and mixing poly(I:C)molecules and JetPEI solutions in a manner that is convenient for thebulk manufacturing of GMP-compliant, stable pharmaceutical formulationsthat comprise poly(I:C)-based complexes with a narrow and controlledsize distribution and that can be then used for producing several vialseach having controlled and comparable features [such as poly(I:C)content, complex stability, and biological activities of theformulations].

Example 2: 1-Vial Process for Production of BO-11X Preparations UnderGMP-Conditions (BO-112 Formulation) Materials & Methods ManufacturingBO-112 Preparations (1-Vial Process)

Poly(I:C) preparations are produced as solutions having concentration,molecule size distribution, and in accordance to the protocol asdescribed in Example 1. However, in one batch, poly(I:C) was itselfobtained by the following exemplary process: poly(C) solution was heatedat 61 to 66° C. for 1.5 h before mixing this with the poly(I) solutionand stirring at 55 to 58° C. for 70 minutes, after which the mixture wascooled and filtered over a 0.2 μm membrane. Some conditions applicableto the chromatography and/or filtration step, absence or presence of afreezing step, together with buffer and annealing time, were adapted forfurther reducing solution viscosity or precipitation of complexes.Either Mannitol or Glucose is used as excipient in the finalformulation. Solution 1 containing JetPEI is obtained by either usingJetPEI in a concentrated liquid preparation or solubilizing solid bulkpreparations of JetPEI (having a molecular weight comprised between 17and 23 kDa) in an amount of sterile water for injection to reach 150 mM,and mixing for obtaining a homogeneous solution. A further dilution stepis performed to reach a concentration of 11.25 mM, before the finaldilution to 5.62 mM in the final vial. Solution 2 contains poly(I:C)molecules and glucose monohydrate in an amount that, after mixing withJetPEI, provides a solution formed by adding 5% glucose (weight/totalvolume of said composition) and poly(I:C) at 0.5-0.7 mg/mL of the totalvolume of said composition, whereby said poly(I:C) complexes with saidJetPEI, thus resulting in a BO-112 composition having from 10⁸ to 10¹⁰particles in solution per vial.

Solution 1 and 2 are independently sterilized using a double filtrationthrough 0.2 μm filters (Sartopore® 2 150 0.2 μm, fully validated assterilizing grade filters (according to ASTM F-838-05 guidelines) usinga pump Watson Marlon (speed 30 rpm). The automated mixing of the twosolutions is performed in each vial using a sequential process: (i)Solution 1 is added to the vial using a Watson-Marlow pump to dose5.95-6.05 g (6 mL; density: 1 g/mL), (ii) Solution 2 is added over thesolution 1 using a 1.8 mm internal diameter tube connected to a G20-0.9μm needle using Flexicon pump at 550 rpm speed to dose 6.08-6.40 g(6mL). Results can be improved by using a T-piece mixer. In case ofaggregates of particles (e.g. with a size in the range of 1-100 μm orlarger) that may be still present by visual inspection at the end of themanufacturing process (or during its storage) due to electrostaticinteractions, the product can be filtered over a 0.8 μm filter prior touse (for instance, before its injection), therewith altering neitherbiological properties nor mono-modal diameter distribution of theparticles within the composition. For example, the BO-112 formulationcan be filtered through a Minisart Syringe Filter (Sartorious) with anexclusion size of 0.8 μm. Vials are sealed with sterile pyrogen-freerubber stoppers and crimp with aluminum capsules and individuallylabelled.

Commercially Available Poly(I:C)-Containing Formulations

Poly-ICLC is a poly(I:C) preparation that is stabilized with polylysineand carboxymethyl-cellulose (Ewel C et al., 1992; WO2005102278).LyoVec-HMW (Cat. No. tlrl-piclv) and LyoVec-LMW (Cat. No. tlrl-picwlv),and corresponding poly(I:C) preparations having high molecular weight(HMW; Cat. Name tlrl-pic) and low molecular weight (LMW; Cat. Nametlrl-picw) are available from Invivogen.

Analytical Tests

The diameter and distribution analysis of BO-112 was performed accordingto Example 1 using standard Dynamic Light Scattering equipment.

Results

The definition of BO-11X preparation applies to the pharmaceuticalcompositions that are obtained by appropriately mixing a solutioncontaining a polymer like PEI with a solution containing poly(I:C)molecules in order to generate complexes having small, narrowlydistributed particle diameter range (as defined by z-average diameterpeaking between 50 and 100 nm and not exceeding 300 nm, or even 200 nm,as shown in Example 1). If BO-111 preparations result from a mixing stepthat is performed manually just before further use (i.e. the “2-vialprocess”), GMP-related and other industrial requirements (e.g. forautomating the process) have been taken into account for establishing a“1-vial process” that provides a BO-11X formulation complying with therequired pharmaceutical specifications and ready to be injected. At thisscope, the two solutions are separately prepared (and, in the case ofpoly(I:C) solution, including also one or more excipient) and sterilizedby filtration before being mixed in automated system wherein speed andtime of mixing are controlled and maintained for each vial.

FIG. 5A provides an overview of such process for generating a first typeof BO-11X preparations that is named BO-112 formulations wherein thedrug substance (i.e. double stranded poly(I:C) molecules that aregenerated by annealing of poly(I) and poly(C) single-stranded molecules)is first mixed with an excipient like glucose in a solution that issterilized by filtration, separately from the solution containing apolymer having the function of carrier (i.e. JetPEI). Then, these twobulk preparations are appropriately mixed into each vial to generate alarge number of structurally and functionally comparable pharmaceuticalformulations that are required for pharmaco-toxicological studies andclinical applications.

This approach takes advantage of the findings described in Example 1,and it can be automated for providing BO-112 preparations with even moreuniform features. This mixing procedure allows not only to incorporateall available Glucose, JetPEI, and poly(I:C) molecules into complexeswithin BO-112 preparations (FIG. 5B) but also to modulate averagediameter and the mono-modal diameter distribution of the complexeswithin BO-112 preparations, so that the Z-average can be modulatedbetween 30 and 150 nm (FIG. 5C). The resulting BO-112 preparationspresent BO-112 complexes having a mono-modal diameter distribution,without visible particles even if the final solution is not filteredthrough 5 μm after mixing the bulk solutions 1 and 2. The mixingconditions can be adapted, in particular by modifying the mixing speedbetween 50 rpm and 600 rpm and/or the flow speed for either poly(I:C) orJetPEI Solution between 1 mL/min and 50 mL/min.

In general, BO-11X preparations (and in particular BO-112 preparations)present the following main features: colorless, no visible particles anosmolarity comprised between 260 and 340 mOsm/kg, a pH comprised between2.7 and 3.4, an optical rotation between +1500 and +3750, a zetapotential equal or superior to 30 mV, a mono-modal diameter distributionof particle with Z-average diameter (nm) between 30 and 150 nm, butpreferably between 60 nm and 130 nm, and comprising poly(I:C) molecules,wherein at least 40% or 50% of such double-stranded polyribonucleotideswith a size higher than 0.85 Kb and at least 70% of such double-strandedpolyribonucleotides have a size comprised between 0.4 and 5 Kb. Featuressuch as diameter distribution of the particles can be modified by usingT-piece mixer in combination with different flow speed for both Solution1 and Solution 2. When such speed is above 20 mL/min (e.g. 30 mL/min),the turbidity of the resulting BO-112 preparation is reduced in parallelwith the reduction of Z-average particle diameter and diameterdistribution around this value, while maintaining mono-modality,possibly due to the change in the flow regime.

The exemplary BO-112 preparation presents a composition similar toBO-111 (formed with 6.924 mg of poly(I:C), 5.625 mM JetPEI, 5% glucose),but each vial comprises particles having a Z-average diameter of between45 +/−5 nm and 81 +/−5 nm (e.g. 73 +/−5 nm), with at least 50% ofparticle smaller than 85 +/−20 nm, the zeta potential of 38 mV, and pH3.1. These structural properties that are maintained after freeze/thawcycle at −20° C. or extensive exposure at room temperature can bemodified in distinct batches, maintaining the acceptance criteria withinspecific ranges of values (for instance, BO-112 formulations can presentan Z-average diameter of 100 +/−50 nm (e.g. 89 nm), with a potential zcomprised between about 40 and 45 mV (e.g. 43 mV) These values may bemodified following cryopreservation but they can still be remain withinthese ranges.

At least some of such reproducibility and acceptance criteria can becompared to the ones of other poly(I:C)-containing formulations forwhich anticancer activity are known. At the level of poly(I:C) moleculessize, the poly(I:C) molecules that are included in the commercialLyovec-HMW and Lyovec-LMW are covering size ranges that are clearlydistinct from those for BO-11X manufacturing process actually makes useof (with HMW almost entirely above 0.85 kb and LMW almost entirely below0.85 kb; FIG. 6A). This size difference in poly(I:C) molecules may bedependent from the different manufacturing process and/or carrier thatare associated in the complexes with poly(I:C) molecules. The Z-averagevalues of complexes within poly(I:C)-based complexes within poly-ICLC(comprising polylysine and carboxymethylcellulose) andLyoVec-HMW/LyoVec-LMW (according to the manufacturer, comprising thecationic lipid-based transfection reagentsDi-tetradecylphoshoryl-N,N,N-trimethylmethanaminium chloride, or DTCPTAand the neutral lipid 1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine,or DiPPE) were compared to the one of BO-112 formulations, showing thatthese commercial formulations contain complexes that are much bigger (inlarge majority larger than 200 nm) and, at least for Lyovec-LMW, with abi-modal distribution (FIG. 6B).

If this analysis is performed after a freeze/thaw cycle, thesecommercial preparations appear also as less stable, with a variabilitynot observed for BO-112 (FIG. 7A-D). Indeed, if BO-112 formulation aZ-average diameter (d· nm) of 100 +/−50 nm (e.g. 82.5 nm), and withoutexceeding 400 nm, as in BO-111 formulations (see FIG. 4 and FIG. 6B),LyoVec-based and Poly-ICLC formulations having a Z-average value wellabove 300 nm, thus confirming that commercially available poly(I:C) areprovided as preparations that are either heterogeneous in composition orinclude large particles that are poorly characterized functionally andwhose size is modified during a freeze/thaw cycle.

Hyperchromicity can be also used to evaluate BO-112 formulation, and inparticular the stability of double-stranded poly(I:C) molecules withinthe particles as a consequence of changes in temperature (or othercondition) determining the separation between poly(I) strands andpoly(C) strands. BO-112 formulation shown a very low hyperchromaticeffect with differences in transmittance at 260 nm lower than 0.2 or0.1. Stability of frozen BO-11X vials at −20° C. for different time hasbeen also assessed and confirmed.

Filtration, lyophilisation and freezing of BO-112 formulation prior toadministration does not promote substantial modifications to thecytotoxic properties, stability, or the structural features on theparticles within the composition with respect to the original BO-112formulation. For instance, compositions maintain D90% below 250 nm, zetapotential between 40 mV and 50 mV, hydrodynamic diameter with aZ-average between 30 and 150 nm, compatibility with the use of glucoseas excipient, polydispersity values comprised between 0.1 and 0.6, andother applicable criteria from European Pharmacopoeia).

Thus, BO-11X preparations, such as BO-112 preparations, are formulationsthat present the high level of stability and reproducibility forparticles formed by poly(I:C)-JetPEI complexes having Z-average diameter(d·nm) below 200 nm (when not below 100 nm) that are not observed forcommercial poly(I:C) formulations that are based on other carriers andmanufacturing methods.

Example 3: Functional Characterization of BO-11X Preparations inCell-Based Models Materials & Methods Poly(I:C) Formulations

The poly(I:C) preparations have been obtained as described in Example 2.

Analysis of Cell Viability

The human melanoma cell line SK-MEL-103 and human pancreatic cancer cellline PANG 02.03 have been used as described in Example 1 and theliterature that is cited herein, using the poly(I:C) formulations atconcentrations that contain poly(I:C) molecules in a range between 0.3and 2.5 μg/mL (0.85 μg/mL being the most relevant reference value) andexposing cells for a period comprised between 12 and 48 hours.

The death-inducing activity of BO-112 was tested in normal melanocytesand cell lines from melanoma and glioblastoma and compared to isolatedcomponents, i.e. poly(I:C) molecules and linear PEI (Jet PEI; Polyplus).Normal melanocytes were isolated from foreskins of asymptomatic donors.Melanoma cells SK-Mel-28, SK-Mel-103, and UACC62 (with mutations in p53,NRAS and BRAF respectively) were obtained from established collectionsat the ATCC or Memorial Sloan Kettering Cancer Centre (USA) and weresubject to short tandem repeat (STR) profiling (GenePrint® 10 System)for cell line authentication. Cells were plated on 96 well plates (6000cells /well). In triplicate per experiment, with. Poly(I:C) only, BO-110or BO-112 formulations at 0.5 or 1 μg/ml for a 24 h or 40 h treatment.

Results

Examples 1 and 2 show experimental data about the initial developmentand characterization of BO-11X formulations, leading to increasedcytotoxicity of BO-11X formulations against cancer cells as compared toBO-110. These data can be further integrated by comparing the BO-11Xmanufacturing process and resulting poly(I:C)-containing preparationswith the processes and preparations that are disclosed in theliterature.

Example 2 has shown that commercial Lyovec-HMW and Lyovec-LMW have asubstantially different distribution. However, it can be evaluated towhich extent the different size of poly(I:C) molecules in thecorresponding poly(I:C) HMW and LMW preparations may also affect theactivity of complexes generated using BO-11X manufacturing process andpoly(I:C) HMW and LMW. If the cytotoxic activity of BO-112 formulationis compared with commercial formulations, the latter ones appear muchless effective in killing cancer cells in at least two in vitro models(FIG. 8A and B). The cytotoxic activity of BO-11X can be measured indifferent types of cancer cell lines, representative of differentclinical cancer indications, to evaluate which cancer indications aremore efficiently treated by BO-11X. These effects may be also studied bymeasuring the expression and/or secretion of proteins that are known tomodify, and possibly improve, the cellular response against cancercells. For instance, a BO-112 formulation induces, much more efficientlythat Poly-ICLC, Interferon-beta expression in a melanoma cell line overa period of at least 24 hours (FIG. 8C). This in vitro evidence can beused for evaluating not only which types of cancer can be moreefficiently treated by administering a BO-11X formulation but also forevaluating which other cancer treatments (such as vaccines, adjuvants,antibodies, chemotherapeutic drugs, radiotherapy, immunotherapy, orinhibitors of enzymatic activities such as kinases) may act in a moreeffective manner when administered in combination with a BO-11Xformulation (e.g. by reducing the dosage, the frequency, and/or theperiod of treatment with this other approach).

Indeed, the specificity of such cytotoxic effects against cancer celllines, and not against normal primary cells, by BO-11X formulations (aspreviously described for lab-scale BO-110 formulations; Tormo D et al.,2009) was confirmed in vitro by comparing the activities withappropriate compounds and cell controls (FIG. 9). Neither linear PEI_(L)nor poly(I:C) molecules alone affect in a significant manner theviability of tumor cells (melanoma or glioma. Only when linear PEI andpoly(I:C) molecules are complexed, a significant killing of tumor cells,without affecting viability of normal melanocytes, is observed. Similarcell-based approaches have been used for validating BO-11X, and BO-112formulation in particular, that have been exposed to filtration,lyophilisation, and/or freezing, confirming that the cytotoxic effectsare qualitatively and quantitatively maintained in BO-112 preparationsafter such processes. More in depth analysis of these in vitro data forguiding clinical development can be performed using differentpre-clinical models involving the production and the comparison ofdifferent BO-11X formulations, different administration regimens, and/orconditions associated to a disease such as cancer.

Example 4: Functional Characterization of BO-11X Preparations in AnimalModels Materials & Methods BO-11X Formulations and Other Compounds

BO-112 formulations have been obtained as described in Example 2, anddiluted with a 5% glucose PBS solution

(vehicle; ref: BE14-516F, Lonza, France) into three differentconcentrations in accordance with a dosing amount per kilo bodyweight ofthe animal of respectively 0.05 mg/Kg, 0.5 mg/Kg and 2.5 mg/Kg.

Murine anti-PD-L1 antibody (InVivoPlus, clone 10F.9G2) was chosen ascombination immunotherapy compound. Each day of injection to mice,anti-PD-1 antibody was diluted with vehicle at final concentrations of1.5 mg/mL.

Results

The anti-cancer, in vivo efficacy of BO-112 formulation was investigatedfor in vivo in an immune competent mouse strain, implanted with mousemelanoma cells. Mice were treated either with a PBS solution or a BO-112formulation at three different concentrations (0.05, 0.5, or 2.5 mg/kg,preferably administered intratumorally), in combination with a murineanti-PD-L1 antibody (preferably administered intravenously) and comparedto the vehicle alone throughout 3 weeks (FIG. 10A). The anti-PD-L1antibody in combination with the vehicle did not significantly increasesurvival when compared to vehicle alone. All three BO-112 formulationscombinations tested with anti-PD-L1 (and possibly independently formsuch antibody) significantly increased survival of the mice compared tovehicle or anti-PD-L1 alone. Moreover, survival significantly increasedin the combination of 2.5 mg/kg BO-112 formulation+anti-PD-L1 comparedto the lower doses of BO-112 formulation. These results correlated withmeasured tumor sizes in these groups.

Anti-PD-L1 antibody is an important and validated anti-cancer drug, andmediator of an effective immune response against the cancer cells. Ourexperiment demonstrates that BO-11X complexes can be used in combinationwith other anti-cancer agents, and that the combination of BO-11Xcompounds with other anti-cancer agents such as anti-PD1 has superiorpotency to the anti-cancer agent alone, leading to significant increasesin survival and anti-tumor efficacy. Moreover, the improvement insurvival correlates with an increase in dose of BO-11X formulation tothe combination, supporting that the added benefit in survival ismediated through the BO-11X formulation.

Thus, BO-11X formulations can be used (alone or in combination withother anti-cancer agents, such as antibodies, immunotherapy, orchemotherapy, that can be administered using the same or a differentroute) in treating melanoma and other cancer indications, in particularthose allowing peritumoral or intratumoral injection such as inpancreatic, endometrial, ovarian, or colorectal cancer. At this scope,the identification of specific biological pathways and mechanisms ofaction may guide the most appropriate dosages, regimens, combinationwith other drugs or therapies, and indications for BO-11X formulations,as shown for combined effects of immunomodulatory monoclonal antibodiestargeting PD-1 or CD137 and poly(I:C) that enhance the activities ofdendritic cells (Sanchez-Paulete A R et al., 2015). To this end, DuewellP et al., 2015 discloses an alternative animal model of disease that maybe used for testing the composition of the present invention.

BO11X therapeutic effect on tumor growth (locally and/or in distallocations) and the anti-tumor immune response can be measured byperforming In vivo studies on Intratumoral (i.t.) administration acrossat a range of concentration for poly(I:C) molecules (such as 0.5, 1, 2,2.5, or 5 mg/kg) to evaluate how such treatment improves mouse survivalin a relevant model, such as a mouse melanoma model, with or withoutco-administering a further drug or a vaccine. At the same time,dose-response studies about specific biological activities induced byBO-11X treatment can be evaluated in parallel ex vivo, using human oranimal samples at the level of apoptosis induction (by Caspase-relatedGlow), chemokine/cytokine secretion in biological fluid (for example,secretion of IL-6 and IP-10), in vitro cellular activation and/orproliferation (for example, associated to CD40, CD86, CD69 upregulationon relevant cell types). These studies can be performed using cells thatare directly involved in disease (e.g. tumor cell, epithelial cell,endothelial or epithelial cells) or indirectly involved since performingsome immune or immunoregulatory activities (e.g. human peripheral bloodmononuclear cells, NK cells, B cells, CD4+/CD8+T cells, dendriticcells). These studies can be further associated to the identification tomolecules that may be used as biomarker to predict response to BO-11X(or lack thereof) in order to stratify disease stages and/or patients'populations for BO-11X treatment.

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1.-25. (canceled)
 26. An aqueous composition comprising one or moreparticles, wherein (a) each particle comprises a complex of at least onedouble-stranded polyribonucleotide, or a salt or solvate thereof, and atleast one polyalkyleneimine, or a salt and/or solvate thereof, wherein(i) the double-stranded polyribonucleotide is polyinosinic-polycytidylicacid [poly(l:C)], wherein at least 60% of the double-strandedpolyribonucleotides have at least 850 base pairs, at least 70% of thedouble-stranded polyribonucleotides have between 400 and 5000 basepairs, and between 20% and 45% of the double-strandedpolyribonucleotides have between 400 and 850 base pairs; and (ii) thepolyalkyleneimine comprises at least 95% linear polyethyleneimines,wherein the average molecular weight of the linear polyalkyleneimine isbetween 17 and 23 kDa and the polydispersity index is <1.5, and whereinthe ratio of the number of moles of nitrogen of the polyalkyleneimine tothe number of moles of phosphorus of the double-strandedpolyribonucleotide in the composition is between 2.5 and 5.5; and (b)the particles have a z-average diameter measured according to ISO 22412of between 30 nm and 150 nm.
 27. The composition according to claim 26,wherein at least 99% of the particles have a mono-modal diameterdistribution below 600 nm.
 28. The composition according to claim 26,wherein the composition has a zeta potential of between 35 and 50 mV.29. The composition according to claim 26, wherein the linearpolyalkyleneimine is a water-soluble homo-polyalkyleneimine or ahetero-polyalkyleneimine.
 30. The composition according to claim 26,wherein the polyribonucleotide composition containspolyinosinic-polycytidylic acid [poly(l:C)] at a concentration of atleast 0.5 mg/ml.
 31. The composition according to claim 26, wherein thecomposition further comprises (a) at least one pharmaceuticallyacceptable carrier, organic solvent, excipient and/or adjuvant; and/or(b) at least one compound selected from an organic compound, aninorganic compound, a nucleic acid, an aptamer, a peptide and a protein.32. The composition according to claim 26, wherein the compositionfurther comprises glucose or mannitol at a concentration of between 1and 10% weight/volume of the composition.
 33. The composition accordingto claim 1, wherein the composition further comprises: (a) a pH ofbetween 2 and 4; and/or (b) an osmolarity of between 200 and 600mOsm/kg.
 34. The composition according to claim 1, wherein thecomposition is formed by making the complex from at least 0.5 mg ofpolyinosinic-polycytidylic acid [poly(l:C)] per ml of the total volumeof the composition.
 35. The composition of claim 26, wherein thecomposition is formed by additionally adding glucose or mannitol in aconcentration of between 1 and 10% weight/volume of the composition. 36.A composition obtained by lyophilisation of the aqueous composition ofclaim
 26. 37. The composition according to claim 26, wherein thecomposition is provided as an injectable, aqueous composition.
 38. Thecomposition according to claim 37, further comprising a pharmaceuticallyacceptable carrier, excipient and/or adjuvant.
 39. The compositionaccording to claim 26 for treating a cell growth disorder characterizedby an abnormal growth of mammalian cells.
 40. The composition accordingto claim 39, wherein the cell growth disorder is cancer or agynecological disorder characterized by an abnormal growth of cells ofthe female mammalian reproductive organs.
 41. The composition for useaccording to claim 37, wherein the composition is injectedintratumorally or peritumorally or injected into skin, an internal organor tissue.
 42. A method for manufacturing the aqueous compositionaccording to claim 26, comprising: (a) providing (i) an aqueous solutionof at least one double-stranded polyribonucleotide, or a salt or solvatethereof, and (ii) an aqueous solution of at least one linearpolyalkyleneimine, or a salt or solvate thereof; (b) filtering eachrespective aqueous solution of step (a) independently through a filterhaving a pore diameter of less than or equal to 500 nm to form arespective sterilized solution; and (c) mixing each respective resultingsterilized solution in a mixing chamber to form the aqueous compositionof claim 26 by simultaneous addition of each respective sterilizedsolution into the mixing chamber, optionally by injection, at a rate ofgreater than or equal to 1 ml/min.
 43. The method according to claim 42,further comprising: (d) filtering the resulting aqueous composition ofstep (c) through a filter having a pore diameter of less than or equalto 600 nm to form a filtrate, or centrifuging the resulting aqueouscomposition at greater than or equal to 22480 m/s² to form asupernatant.
 44. The method of claim 43, further comprising: (e)lyophilising the resulting aqueous composition, filtrate or supernatant.45. The method according to claim 42, wherein step (c) comprises mixingeach respective resulting sterilized solution in a mixing chamber toform the aqueous composition by sequential addition of one sterilizedsolution into the other sterilized solution in the mixing chamber,optionally by injection, at a rate of greater than or equal to 1 ml/min.46. The method according to claim 41, wherein each of or both aqueoussolutions of step (a) further comprise a pharmaceutically acceptablecarrier, organic solvent, excipient and/or adjuvant.